Aptamer therapeutics useful in the treatment of complement-related disorders

ABSTRACT

The invention provides nucleic acid therapeutics and methods for using these nucleic acid therapeutics in the treatment of complement-related disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/289,374, filed Feb. 28, 2019, which is a continuation of U.S.application Ser. No. 15/448,238, which is a continuation of U.S.application Ser. No. 14/573,423, filed Dec. 17, 2014, now U.S. Pat. No.9,617,546, which is a continuation of U.S. application Ser. No.13/783,633, filed Mar. 4, 2013, now U.S. Pat. No. 8,946,184, which is acontinuation of U.S. application Ser. No. 13/525,680, filed Jun. 18,2012, now U.S. Pat. No. 8,436,164, which is a division of U.S.application Ser. No. 11/884,411, filed Aug. 26, 2008, now U.S. Pat. No.8,236,773, which is the U.S. national stage of International ApplicationNo. PCT/US2006/005215, filed Feb. 14, 2006, which is acontinuation-in-part of U.S. application Ser. No. 11/058,134, filed Feb.14, 2005, now U.S. Pat. No. 7,803,931, the disclosure of each of whichis incorporated by reference herein in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:OPHT_005_11US_SeqList_ST25.txt, date recorded: Feb. 28, 2017, file size73 kilobytes).

FIELD OF THE INVENTION

The invention relates generally to the field of nucleic acids and moreparticularly to aptamers capable of binding to the C5 protein of thecomplement system, useful as therapeutics in and diagnostics incomplement-related cardiac, inflammatory, and auto-immune disorders,ischemic reperfusion injury and/or other diseases or disorders in whichC5 mediated complement activation has been implicated. The inventionfurther relates to materials and methods for the administration ofaptamers capable of binding to the C5 complement system protein.

BACKGROUND OF THE INVENTION

Aptamers are nucleic acid molecules having specific binding affinity tomolecules through interactions other than classic Watson-Crick basepairing.

Aptamers, like peptides generated by phage display or monoclonalantibodies (“MAbs”), are capable of specifically binding to selectedtargets and modulating the target's activity, e.g., through bindingaptamers may block their target's ability to function. Created by an invitro selection process from pools of random sequence oligonucleotides,aptamers have been generated for over 100 proteins including growthfactors, transcription factors, enzymes, immunoglobulins, and receptors.A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds itstarget with sub-nanomolar affinity, and discriminates against closelyrelated targets (e.g., aptamers will typically not bind other proteinsfrom the same gene family). A series of structural studies have shownthat aptamers are capable of using the same types of bindinginteractions (e.g., hydrogen bonding, electrostatic complementarity,hydrophobic contacts, steric exclusion) that drive affinity andspecificity in antibody-antigen complexes.

Aptamers have a number of desirable characteristics for use astherapeutics and diagnostics including high specificity and affinity,biological efficacy, and excellent pharmacokinetic properties. Inaddition, they offer specific competitive advantages over antibodies andother protein biologics, for example:

1) Speed and control. Aptamers are produced by an entirely in vitroprocess, allowing for the rapid generation of initial leads, includingtherapeutic leads. In vitro selection allows the specificity andaffinity of the aptamer to be tightly controlled and allows thegeneration of leads, including leads against both toxic andnon-immunogenic targets.

2) Toxicity and Immunogenicity. Aptamers as a class have demonstratedlittle or no toxicity or immunogenicity. In chronic dosing of rats orwoodchucks with high levels of aptamer (10 mg/kg daily for 90 days), notoxicity is observed by any clinical, cellular, or biochemical measure.Whereas the efficacy of many monoclonal antibodies can be severelylimited by immune response to antibodies themselves, it is extremelydifficult to elicit antibodies to aptamers most likely because aptamerscannot be presented by T-cells via the MHC and the immune response isgenerally trained not to recognize nucleic acid fragments.

3) Administration. Whereas most currently approved antibody therapeuticsare administered by intravenous infusion (typically over 2-4 hours),aptamers can be administered by subcutaneous injection (aptamerbioavailability via subcutaneous administration is >80% in monkeystudies (Tucker et al., J. Chromatography B. 732: 203-212, 1999)). Thisdifference is primarily due to the comparatively low solubility and thuslarge volumes necessary for most therapeutic MAbs. With good solubility(>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa;antibody: 150 kDa), a weekly dose of aptamer may be delivered byinjection in a volume of less than 0.5 mL. In addition, the small sizeof aptamers allows them to penetrate into areas of conformationalconstrictions that do not allow for antibodies or antibody fragments topenetrate, presenting yet another advantage of aptamer-basedtherapeutics or prophylaxis.

4) Scalability and cost. Therapeutic aptamers are chemically synthesizedand consequently can be readily scaled as needed to meet productiondemand. Whereas difficulties in scaling production are currentlylimiting the availability of some biologics and the capital cost of alarge-scale protein production plant is enormous, a single large-scaleoligonucleotide synthesizer can produce upwards of 100 kg/year andrequires a relatively modest initial investment. The current cost ofgoods for aptamer synthesis at the kilogram scale is estimated at$500/g, comparable to that for highly optimized antibodies. Continuingimprovements in process development are expected to lower the cost ofgoods to <$100/g in five years.

5) Stability. Therapeutic aptamers are chemically robust. They areintrinsically adapted to regain activity following exposure to factorssuch as heat and denaturants and can be stored for extended periods (>1yr) at room temperature as lyophilized powders.

The Complement System

The complement system comprises a set of at least 20 plasma and membraneproteins that act together in a regulated cascade system to attackextracellular forms of pathogens (e.g., bacterium). The complementsystem includes two distinct enzymatic activation cascades, theclassical and alternative pathways (FIG. 1), and a non-enzymatic pathwayknown as the membrane attack pathway.

The first enzymatically activated cascade, known as the classicalpathway, comprises several components, C1, C4, C2, C3 and C5 (listed byorder in the pathway). Initiation of the classical pathway of thecomplement system occurs following binding and activation of the firstcomplement component (C1) by both immune and non-immune activators. C1comprises a calcium-dependent complex of components C1q, C1r and C1s,and is activated through binding of the C1q component. C1q contains sixidentical subunits and each subunit comprises three chains (the A, B andC chains). Each chain has a globular head region that is connected to acollagen-like tail. Binding and activation of C1q by antigen-antibodycomplexes occurs through the C1q head group region. Numerousnon-antibody C1q activators, including proteins, lipids and nucleicacids, bind and activate C1q through a distinct site on thecollagen-like stalk region. The C1qrs complex then catalyzes theactivation of complement components C4 and C2, forming the C4bC2acomplex which functions as a C3 convertase.

The second enzymatically activated cascade, known as the alternativepathway, is a rapid, antibody-independent route for complement systemactivation and amplification. The alternative pathway comprises severalcomponents, C3, Factor B, and Factor D (listed by order in the pathway).Activation of the alternative pathway occurs when C3b, a proteolyticcleavage form of C3, is bound to an activating surface agent such as abacterium. Factor B is then bound to C3b, and cleaved by Factor D toyield the active enzyme, Ba. The enzyme Ba then cleaves more C3 togenerate more C3b, producing extensive deposition of C3b-Ba complexes onthe activating surface.

Thus, both the classical and alternate complement pathways produce C3convertases that split factor C3 into C3a and C3b. At this point, bothC3 convertases further assemble into C5 convertases (C4b2a3b andC3b3bBb). These complexes subsequently cleave complement component C5into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide(170 kDa). The C5a polypeptide binds to a 7 transmembrane G-proteincoupled receptor, which was originally associated with leukocytes and isnow known to be expressed on a variety of tissues including hepatocytesand neurons. The C5a molecule is the primary chemotactic component ofthe human complement system and can trigger a variety of biologicalresponses including leukocyte chemotaxis, smooth muscle contraction,activation of intracellular signal transduction pathways,neutrophil-endothelial adhesion, cytokine and lipid mediator release andoxidant formation.

The larger C5b fragment binds sequentially to later components of thecomplement cascade, C6, C7, C8 and C9 to form the C5b-9 membrane attackcomplex (“MAC”). The C5b-9 MAC can directly lyse erythrocytes, and ingreater quantities, it is lytic for leukocytes and damaging to tissuessuch as muscle, epithelial and endothelial cells. In sublytic amounts,the MAC can stimulate upregulation of adhesion molecules, intracellularcalcium increase and cytokine release. In addition, the C5b-9 MAC canstimulate cells such as endothelial cells and platelets without causingcell lysis. The non-lytic effects of C5a and the C5b-9 MAC are sometimesquite similar.

Although the complement system has an important role in the maintenanceof health, it has the potential to cause or contribute to disease. Forexample, the complement system has been implicated in side effectsrelating to coronary artery bypass graft (“CABG”) surgery, numerousrenal, rheumatological, neurological, dermatological, hematological,vascular/pulmonary, allergy, infectious, and biocompatibility/shockdiseases and/or conditions, and diabetic retinopathy. The complementsystem is not necessarily the only cause of a disease state, but it maybe one of several factors that contribute to pathogenesis.

In Fitch et al., Circ. 100:2499-506 (1999), the effects of the anti-C5single-chain antibody fragment Pexelizumab on patients undergoingcoronary artery bypass graft surgery with cardiopulmonary bypass (“CPB”)was tested. Individual patients were administered Pexelizumab in a 10minute, single-bolus dose just prior to CPB at 0.5 mg/kg, 1.0 mg/kg and2.0 mg/kg. Blood samples were removed and tested for complement activityat pre-dose, 5 min post-dose, after 5 min at 28° C., after initiation ofrewarming, after 5 min at 37° C., and up to 7 days after CPB.Pharmacodynamic analysis demonstrated significant dose-dependentinhibition of complement hemolytic activity for up to 14 hours at adosage of 2 mg/kg, and the generation of proinflammatory complementbyproducts (sC5b-9) was effectively inhibited in a dose-dependentfashion. As previously mentioned, however, antibody therapeutics havecertain limitations.

Accordingly, it would be beneficial to have novel inhibitors of thecomplement system for use as therapeutics and diagnostics in thetreatment of complement-related disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration depicting the classical and alternativepathways of the complement system.

FIG. 2 is a schematic representation of the in vitro aptamer selection(SELEX™) process from pools of random sequence oligonucleotides.

FIG. 3A is an illustration depicting the nucleotide sequence andsecondary structure of an anti-C5 aptamer (SEQ ID NO: 1), in which theunderlined residues are either 2′-H pyrimidine residues or 2′-fluoropyrimidine residues, the boxed residues are either 2′-fluoro pyrimidineresidues or 2′-OMe pyrimidine residues, and the residues indicated by anarrow (→) represent residues that must contain a 2′-fluoro modification.

FIG. 3B is an illustration depicting the nucleotide sequence andsecondary structure of the ARC330 anti-C5 aptamer (SEQ ID NO: 2), inwhich the circled residues are 2′-H residues, the pyrimidine residuesare 2′-fluoro substituted, and the majority of purine residues are2′-OMe substituted, except for the three 2′-OH purine residues shown inoutline.

FIG. 3C is an illustration depicting the nucleotide sequence andsecondary structure of the ARC186 anti-C5 aptamer (SEQ ID NO: 4) inwhich all 21 pyrimidine residues have 2′-fluoro modifications and themajority of purines (14 residues) have 2′-OMe modifications, except forthe three 2′-OH purine residues shown in outline.

FIG. 4 is an illustration of a 40 kD branched PEG (1,3-bis(mPEG-[20kDa])-propyl-2-(4′-butamide).

FIG. 5 is an illustration of a 40 kD branched PEG (1,3-bis(mPEG-[20kDa]) propyl-2-(4′-butamide) attached to the 5′end of an aptamer.

FIG. 6 is an illustration depicting various strategies for synthesis ofhigh molecular weight PEG-nucleic acid conjugates.

FIG. 7A is a graph comparing dose dependent inhibition of hemolysis byPEGylated anti-C5 aptamers (ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO:62), and ARC187 (SEQ ID NO: 5)), to a non-PEGylated anti-C5 aptamer(ARC186 (SEQ ID NO: 4)); FIG. 7B is a table of the IC₅₀ values of theaptamers used in the hemolysis assay depicted in FIG. 7A; FIG. 7C is agraph comparing dose dependent inhibition of hemolysis by PEGylatedanti-C5 aptamers ARC187 (SEQ ID NO: 5), ARC1537 (SEQ ID NO: 65), ARC1730(SEQ ID NO: (66), and ARC1905 (SEQ ID NO: 67); FIG. 7D is a table of theIC₅₀ values of the aptamers used in the hemolysis assay depicted in FIG.7C.

FIG. 8 is a graph of percent inhibition of hemolysis by the anti-C5aptamer, ARC658 (SEQ ID NO: 62), of cynomolgus serum complement versushuman serum complement.

FIG. 9 is a graph depicting the binding of ARC186 (SEQ ID NO: 4) topurified C5 protein at both 37° C. and room temperature (23° C.)following a 15 minute incubation.

FIG. 10 is another graph depicting the binding of ARC186 (SEQ ID NO: 4)to purified C5 protein at both 37° C. and room temperature (23° C.)following a 4 hour incubation.

FIG. 11 is graph showing a time course of dissociation of C5⋅ARC186complex at 23° C.

FIG. 12 is a graph showing a time course of equilibration in theformation of C5⋅ARC186 complex at 23° C.

FIG. 13 is a graph depicting ARC186 (SEQ ID NO: 4) binding to C5 proteinversus protein components upstream and downstream in the complementcascade.

FIG. 14 is a graph depicting the percentage of radiolabeled ARC186 (SEQID NO: 4) that bound C5 in the presence of unlabeled competitor ARC186(SEQ ID NO: 4), ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO: 62) or ARC187(SEQ ID NO: 5).

FIG. 15 is a graph depicting the amount of C5b complement proteinproduced in blood samples incubated for 5 hours at 25° C. and 37° C. inthe presence of varying concentrations of the ARC186 (SEQ ID NO: 4)aptamer.

FIG. 16 is a graph depicting percent complement inhibition by ARC 187(SEQ ID NO: 5) in the presence of zymosan in undiluted human serum,citrated human whole blood or cynomolgus serum.

FIG. 17 is a graph showing ARC658 (SEQ ID NO: 62) frilly inhibitscomplement activation (C5a) in the tubing loop model described inExample 1D.

FIG. 18 is a graph depicting the dissociation constants for Round 10 ofthe C5 selection pools. Dissociation constants (K_(d)s) were estimatedby fitting the data to the equation: fraction RNAbound=amplitude*K_(d)/(K_(d)+[C5]). “ARC520” (SEQ ID NO: 70) refers tothe naïve unselected dRmY pool and the “+” indicates the presence ofcompetitor (0.1 mg/ml tRNA, 0.1 mg/ml salmon sperm DNA).

FIG. 19 is a graph depicting C5 clone dissociation constant curves.Dissociation constants (K_(d)s) were estimated by fitting the data tothe equation: fraction RNA bound=amplitude*K_(d)/(K_(d)+[C5]).

FIG. 20 is a graph depicting an IC₅₀ curve that illustrates theinhibitory effect on hemolysis activity of varying concentrations ofanti-C5 aptamer clone ARC913 (SEQ ID NO: 75) as compared to ARC186 (SEQID NO: 4).

FIG. 21 is an illustration depicting the structure of ARC 187 (SEQ IDNO: 5).

FIG. 22 is an illustration depicting the structure of ARC1905 (SEQ IDNO: 67).

FIG. 23 is a table outlining the experimental design of the firstisolated perfused heart study.

FIG. 24 is a graph comparing the pressure tracings for theintraventricular pressure in the left ventricle (LV) of an isolatedheart exposed to human plasma with the LVP pressure tracings of anisolated heart exposed to the control aptamer solution.

FIG. 25 is a graph comparing the pressure tracings for theintraventricular pressure in the left ventricle (LV) of the isolatedhearts exposed to the molar equivalent, 10× and 50× aptamer/C5 solutions(where a concentration of approximately 500 nM is assumed for C5 innormal, undiluted human plasma).

FIG. 26 is a graph comparing the heart rate changes in beats per minute(bpm) in isolated mouse hearts after exposure to human plasma andvarious plasma/aptamer solutions.

FIG. 27 is a graph comparing the changes in the heart weight in isolatedmouse hearts before and after exposure to human plasma containing 0-1×molar ratio ARC186 (SEQ ID NO: 4) (failed hearts), or 10-50× molar ratio(hearts protected with C5 aptamer).

FIG. 28 is a graph comparing the relative C5a production in humanplasma, containing varying aptamer concentrations, following perfusionthrough isolated mouse hearts. Relative C5a concentrations are plottedas absorbance units (Abs), where higher readings reflect the presence ofhigher C5a levels.

FIG. 29 is a graph comparing the relative soluble C5b-9 production inhuman plasma containing varying aptamer concentrations, followingperfusion through isolated mouse hearts.

FIG. 30 is a graph showing the effect of ARC 186 (SEQ ID NO: 4) on C3cleavage in mouse heart effluent.

FIG. 31 is a table showing the immunohistochemistry staining results forthe isolated perfused mouse heart study.

FIG. 32 is a table showing the molar ratio of ARC658 (SEQ ID NO: 62)necessary, in human or primate serum, to protect the heart fromC5b-mediated damage.

FIG. 33 is a graph showing a log-linear plot of remaining percent offull-length ARC 186 as a function of incubation time in both rat andcynomolgus macaque plasma.

FIG. 34 is a table showing the experimental design of thepharmacokinetic study conducted Sprague-Dawley rats as described inExample 5.

FIG. 35 is a table showing mean plasma concentration of ARC657 (SEQ IDNO: 61), ARC658 (SEQ ID NO: 62) or ARC187 (SEQ ID NO: 5) versus time inSprague-Dawley rats.

FIG. 36 is a graph depicting mean plasma concentration of ARC657 (SEQ IDNO: 61), ARC658 (SEQ ID NO: 62) and ARC187 (SEQ ID NO: 5) over timefollowing intravenous administration of aptamer in rats.

FIG. 37 is a table showing the noncompartmental analysis of theconcentration versus time data depicted in FIG. 35 and FIG. 36.

FIG. 38A is a table showing the design for the pharmacokinetic study ofARC187 (SEQ ID NO: 5) and ARC1905 (SEQ ID NO: 67) in mice; FIG. 38B is agraph depicting the pharmacokinetic profile of ARC187 (SEQ ID NO: 5) andARC1905 (SEQ ID NO: 67) in CD-1 mice after a single IV bolusadministration; FIG. 38C is a table showing the noncompartmentalanalysis of the concentration versus time data depicted in FIG. 38B.

FIG. 39 is a table showing detection of the listed aptamers in mouseheart tissue following intravenous administration.

FIG. 40 is a table showing the experimental design of animal Study 1,described in Example 5E.

FIG. 41 is a table showing aptamer plasma concentration versus timefollowing intravenous bolus administration of aptamer to cynomolgusmacaques.

FIG. 42 is a table listing the pharmacokinetic parameters for ARC657(SEQ ID NO: 61), ARC658 (SEQ ID NO: 62) and ARC 187 (SEQ ID NO: 5)administered intravenously to cynomolgus macaque in Study 1.

FIG. 43A and FIG. 43C are graphs depicting plasma concentrations ofsC5b-9 and C5a over time following intravenous administration of theanti-C5 aptamers ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO: 62), orARC187 (SEQ ID NO: 5) to cynomolgus macaques;

FIG. 43B and FIG. 43D are graphs depicting plasma concentrations ofsC5b-9 and C5a versus concentration of anti-C5 aptamers, ARC657 (SEQ IDNO: 61), ARC658 (SEQ ID NO: 62), or ARC187 (SEQ ID NO: 5).

FIG. 44 is a table showing the experimental design of Study 2, describedin Example 5F.

FIG. 45 is a graph showing the mean aptamer plasma concentration atvarious time points following intravenous administration of ARC658 (SEQID NO: 62), or ARC187 (SEQ ID NO: 5) to cynomolgus macaques.

FIG. 46 is a table showing the two compartmental analysis of theconcentration versus time data following intravenous bolus aptameradministration to cynomolgus macaque.

FIG. 47 is a graph depicting C5b-9 concentration versus ARC 187 (SEQ IDNO: 5) or ARC658 (SEQ ID NO: 62) concentration in the presence ofzymosan in cynomolgus plasma.

FIG. 48 is a graph depicting C5a concentration versus ARC187 (SEQ ID NO:5) or ARC658 (SEQ ID NO: 62) concentration in the presence of zymosan incynomolgus plasma.

FIG. 49 is a table summarizing the PK-PD study of ARC187 (SEQ ID NO: 5)during and after IV bolus plus infusion administration to cynomolgusmacaques.

FIG. 50 is a table summarizing the pharmacokinetic parameters for ARC187 (SEQ ID NO: 5) in cynomolgus macaques after IV bolus administration.

FIG. 51 is a graph depicting the calculated and actual measuredpharmacokinetic profiles of ARC187 (SEQ ID NO: 5) during and after IVbolus plus infusion administration to cynomolgus macaques.

FIG. 52 is a graph showing the plasma levels of active ARC187 (SEQ IDNO: 5) remain constant during and after IV bolus plus infusionadministration to cynomolgus macaques.

FIG. 53 is a table showing the predicted human dosing requirements foranti-C5 aptamers in CABG surgery.

FIG. 54 is a graph depicting ARC187 (SEQ ID NO: 5) has relatively no invitro effect on coagulation as measured by the prothrombin time (PT) andactivated partial thromboplastin time (APTT).

FIG. 55 is a table summarizing the in vitro effects of ARC 187 (SEQ IDNO: 5) on anti-coagulation activity of heparin, and procoagulationactivity of protamine.

FIG. 56 is a graph showing ARC 187 (SEQ ID NO: 5) does not effect thereversal of heparin anticoagulation in vivo.

FIG. 57 is graph showing heparin and protamine both have no effect onARC187 (SEQ ID NO: 5) anti-complement function, measured by inhibitionof complement activation of zymosan.

FIG. 58 is a graph depicting the percent inhibition of sheep erythrocytehemolysis in the presence of human serum as a function of concentrationof anti-C5 aptamers ARC1905 (SEQ ID NO 67) or ARC672 (SEQ ID NO 63).

FIG. 59A is a graph depicting the percent inhibition of hemolysis in thepresence of human, cynomolgus monkey and rat serum by ARC1905 (SEQ ID NO67); FIG. 59B is a table summarizing the mean IC₅₀ values for inhibitionof complement activation in human, cynomolgus monkey and rat serum byARC1905, an anti-C5 aptamer or ARC127, an irrelevant aptamer which doesnot bind C5 (negative control).

FIG. 60 is a graph depicting the IC₅₀ value for inhibition ofradiolabeled ARC186 (SEQ ID NO: 4) (vertical axis) as a function ofconcentration of unlabeled competitor ARC1905 (SEQ ID NO 67) or ARC672(SEQ ID NO 63) (horizontal axis), in a competition binding assay.

FIG. 61 is a graph depicting the IC₅₀ value for inhibition ofradiolabeled ARC186 (SEQ ID NO: 4) (vertical axis) as a function ofconcentration of unlabeled competitor ARC1905 (SEQ ID NO 67) (horizontalaxis) at 37° C. and 25° C. in a competition binding assay.

FIG. 62 is a graph depicting standard curves for human C5a (hC5a) andcynomolgus monkey C5a (hC5a eq).

FIG. 63 is a table summarizing the IC₅₀, IC₉₀ and IC₉₉ values forinhibition of C5 activation in human and cynomolgus monkey serum byARC1905 (SEQ ID NO 67), as measured in a zymosan-induced complementactivation assay.

FIG. 64 is a graph depicting the percent inhibition of C5a generation asa function of ARC1905 (SEQ ID NO 67) concentration in human andcynomolgus monkey sera as measured in a zymosan-induced complementactivation assay.

FIG. 65 is a graph depicting the effect of ARC1905 (SEQ ID NO 67) on C3ageneration in human or cynomolgus monkey serum, as measured in azymosan-induced complement activation assay.

FIG. 66 is a table summarizing the mean IC₅₀, IC₉₀ and IC₉₉ values forARC1905 inhibition of complement activation (SEQ ID NO 67) in humanserum from 5 donors, as measured in a tubing loop model of complementactivation.

FIG. 67 is a graph depicting the percent inhibition of C5a and C3ageneration as a function of concentration of ARC1905, an anti-C5aptamer, or ARC127, an irrelevant aptamer which does not bind C5(negative control) in a tubing loop model of complement activation.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for the treatment,prevention and amelioration of complement related disease. In oneembodiment, an aptamer comprising a nucleotide sequence according toARC186 (SEQ ID NO: 4) conjugated to a PEG moiety is provided. Inparticular embodiments, this ARC186 aptamer/PEG conjugate comprisessubstantially the same binding affinity for C5 complement protein as anaptamer consisting of the sequence according to SEQ ID NO: 4 but lackingthe PEG moiety. Substantially the same binding affinity as used hereinmeans no more than about a 2 to ten fold difference, preferably no morethan a 2 to five fold difference in dissociation constants as measuredby dot blot analysis. In some embodiments the dissociation constants aremeasured by competition dot blot analysis as described in Example 1Abelow. In some embodiments, the polyethylene glycol moiety comprises amolecular weight greater than 10 kDA, particularly a molecular weight of20 kDA, more particularly 30 kDa and more particularly 40 kDa. In someembodiments, the PEG moiety is conjugated to the 5′ end of ARC186 (SEQID NO:4). In some embodiments the aptamer/PEG conjugate comprises a halflife, preferably the terminal half life in a two compartment model asdetermined by the method described in Example 5E below, of at least 15hours, preferably at least 24 hours, more preferably at least 48 hoursin primate. In some embodiments the aptamer/PEG conjugate comprises ahalf life, preferably the terminal half life in a two compartment model,of at least 10, preferably at least 15 hours in rat. In someembodiments, the PEG conjugated to the 5′ end of ARC186 (SEQ ID NO: 4)is a 40 kDa PEG. In particular embodiments the 40 kDa PEG is a branchedPEG. In some embodiments the branched 40 kDa PEG is 1,3-bis(mPEG-[20kDa])-propyl-2-(4′-butamide). In other embodiments the branched 40 kDaPEG is 2,3-bis(mPEG-[20 kDa])-propyl-1-carbamoyl.

In embodiments where the branched 40 kDa PEG is 1,3-bis(mPEG-[20kDa])-propyl-2-(4′-butamide), an aptamer having the structure set forthbelow is provided:

where,

-   -   indicates a linker    -   Aptamer=fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfU        fUAfCfCfUmGfCmG-3T (SEQ ID NO: 4),        -   wherein fC and fU=2′-fluoro nucleotides, and mG and            mA=2′-OMe nucleotides and all other nucleotides are 2′-OH            and 3T indicates an inverted deoxy thymidine.

In embodiments where the branched 40 kDa PEG is 2,3-bis(mPEG-[20kDa])-propyl-1-carbamoyl, an aptamer having the structure set forthbelow is provided:

where,

-   -   indicates a linker    -   Aptamer=fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfU        fUAfCfCfUmGfCmG-3T (SEQ ID NO: 4),    -   wherein fC and fU=2′-fluoro nucleotides, and mG and mA=2′-OMe        nucleotides and all other nucleotides are 2′-OH and 3T indicates        an inverted deoxy thymidine.

In some embodiments of this aspect of the invention the linker is analkyl linker. In particular embodiments, the alkyl linker comprises 2 to18 consecutive CH2 groups. In preferred embodiments, the alkyl linkercomprises 2 to 12 consecutive CH2 groups. In particularly preferredembodiments the alkyl linker comprises 3 to 6 consecutive CH2 groups.

In a particular embodiment an aptamer, ARC187 (SEQ ID NO: 5), having thestructure set forth below is provided:

-   -   where        Aptamer=fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfU        fUAfCfCfUmGfCmG-3T (SEQ ID NO: 4)        -   wherein fC and fU=2′-fluoro nucleotides, and mG and            mA=2′-OMe nucleotides and all other nucleotides are 2′-OH            and where 3T indicates an inverted deoxy thymidine.

In another embodiment an aptamer, ARC1905 (SEQ ID NO: 67), having thestructure set forth below is provided:

-   -   where        Aptamer=fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCtUmGmAmGfUfCfUmGmAmGfUfU        fUAfCfCfUmGfCmG-3T (SEQ ID NO: 4)        -   wherein fC and fU=2′-fluoro nucleotides, and mG and            mA=2′-OMe nucleotides and all other nucleotides are 2′-OH            and where 3T indicates and inverted deoxy thymidine.

In another aspect, the invention provides pharmaceutical compositions.In one embodiment, a pharmaceutical composition comprising atherapeutically effective amount of ARC187 (SEQ ID NO: 5) or ARC1905(SEQ ID NO: 67) or a salt thereof is provided. The pharmaceuticalcomposition of the invention may comprise a pharmaceutically acceptablecarrier or diluent. In this aspect, the invention provides apharmaceutical composition comprising a therapeutically effective amountof an aptamer that inhibits C5 complement protein cleavage in vivo or asalt thereof and a pharmaceutically acceptable carrier or diluent. Inthis aspect of the invention an ARC187 (SEQ ID NO: 5) or ARC1905 (SEQ IDNO: 67) pharmaceutical composition for use in the treatment, preventionor amelioration of disease in vivo is provided. Also, in this aspect ofthe invention ARC187 (SEQ ID NO: 5) or ARC1905 (SEQ ID NO: 67) for theuse in the preparation of a pharmaceutical composition are provided.

In another aspect of the invention, methods of treatment are provided.In one embodiment, the method of the invention comprises treating,preventing or ameliorating a disease mediated by C5 complement protein,and/or it's derivatives C5a and C5b-9, the method includingadministering a pharmaceutical composition comprising ARC187 (SEQ ID NO:5) or ARC1905 (SEQ ID NO: 67) or a salt thereof to a vertebrate. In someembodiments, the method comprises administering the pharmaceuticalcomposition of the invention to a mammal. In some embodiments, themammal is a human.

In some embodiments, the C5 complement protein, C5a and/orC5b-9-mediated disease to be treated is acute ischemic diseases(myocardial infarction, stroke, ischemic/reperfusion injury); acuteinflammatory diseases (infectious disease, septicemia, shock,acute/hyperacute transplant rejection); chronic inflammatory and/orimmune-mediated diseases (allergy, asthma, rheumatoid arthritis, andother rheumatological diseases, multiple sclerosis and otherneurological diseases, psoriasis and other dermatological diseases,myasthenia gravis, systemic lupus erythematosus (SLE), subacute/chronictransplant rejection, glomerulonephritis and other renal diseases). Insome embodiments, the C5 complement protein, C5a and/or C5b-9 mediateddiseases to be treated include complement activation associated withdialysis or circumstances in which blood is passed over and/or throughsynthetic tubing and/or foreign material. In some embodiments, the C5complement protein, C5a and/or C5b-9-mediated disease to be treated isselected from the group consisting of myocardial injury relating to CABGsurgery, myocardial injury relating to balloon angioplasty andmyocardial injury relating to restenosis. In some embodiments, C5complement protein, C5a and/or C5b-9-mediated disorder to be treated isselected from the group consisting of: myocardial injury relating toCABG surgery, myocardial injury relating to balloon angioplasty,myocardial injury relating to restenosis, complement protein mediatedcomplications relating to CABG surgery, complement protein mediatedcomplications relating to percutaneous coronary intervention,paroxysomal nocturnal hemoglobinuria, acute transplant rejection,hyperacute transplant rejection, subacute transplant rejection, andchronic transplant rejection. In some embodiments the C5 complementprotein C5a and/or C5b-9-mediated disease to be treated is complicationsrelating to CABG surgery. In a particular embodiment, the disease to betreated is myocardial injury relating to CABG surgery

In some embodiments, the method of the invention includes administeringthe pharmaceutical composition comprising ARC187 (SEQ ID NO: 5) orARC1905 (SEQ ID NO: 67) to achieve an aptamer plasma concentration thatis about 0.5 to about 10 times that of the endogenous C5 complementprotein. In some embodiments, the pharmaceutical ARC187 (SEQ ID NO: 5)or ARC 1905 (SEQ ID NO: 67) aptamer compositions are administered toachieve an aptamer plasma concentration that is about 0.75 to about 5times, 0.75 to about 3 times, and 1.5 to about 2 times that of theendogenous C5 complement protein while in other embodiments the aptamercomposition is administered to achieve a concentration equivalent tothat of the endogenous complement protein. In some embodiments, thepharmaceutical composition of the invention comprising ARC187 (SEQ IDNO: 5) or ARC 1905 (SEQ ID NO: 67) is administered to achieve an aptamerplasma concentration of about 5 μM, about 4 μM, about 3 μM, about 2 μM,about 1.5 μM, about 1 μM or of about 500 nM.

Any combination of route, duration, and rate of administration may beused that is sufficient to achieve the aptamer plasma concentrations ofthe invention. In some embodiments the pharmaceutical composition isadministered intravenously. In some embodiments, the pharmaceuticalcomposition is administered as a bolus and/or via continuous infusion.

In particular embodiments of treating, preventing and/or amelioratingcomplications related to CABG surgery, particularly myocardial injuryrelated to CABG surgery, the method of the invention comprisesadministering the pharmaceutical composition prior to surgery andcontinuing administration at least 24 hours, in some embodiments about48 hours or in some embodiments about 72 hours. In a particularembodiment of this aspect of the invention, a plasma aptamerconcentration of about two times the endogenous complement proteinconcentration is achieved by administration of an intravenous bolus ofabout 0.75 to 1.25, preferably of about 1 mg of aptamer per kg of thepatient to be treated in advance of, simultaneously with or afterintravenous infusion of a lower dose of aptamer wherein mg does notinclude the weight of the conjugated PEG. In some embodiments the lowerdose will be infused at a rate selected from the range of 0.001 to 0.005mg/kg/min wherein mg does not include the weight of the conjugated PEG.In a particular embodiment, the lower dose will be infused at a rate ofabout 0.0013 mg/kg/min. In still other embodiments of this aspect of theinvention, where the aptamer/conjugate comprises a sufficiently longhalf life, the aptamer pharmaceutical composition may be administeredonce or twice daily as an intravenous bolus dose.

In another aspect of the invention, diagnostic methods are provided. Inone embodiment, the diagnostic method comprises contacting the ARC187(SEQ ID NO: 5) or ARC 1905 (SEQ ID NO: 67) with a composition suspectedof comprising C5 complement protein or a variant thereof, and detectingthe presence or absence of C5 complement protein or a variant thereof.In some embodiments the complement protein or variant are vertebrate,particularly mammalian, and more particularly human. The presentinvention provides an ARC187 (SEQ ID NO: 5) or ARC1905 (SEQ ID NO: 67)composition for use as an in vitro or in vivo diagnostic.

In another aspect of the invention, an aptamer comprising a nucleotidesequence selected from the group consisting of: ARC 330 (SEQ ID NO: 2)and ARC188-189, ARC250, ARC296-297, ARC331-334, ARC411-440, ARC457-459,ARC473, ARC522-525, ARC532, ARC543-544, ARC550-554, ARC657-658, ARC672,ARC706, ARC1537, ARC1730, (SEQ ID NOS: 6 to SEQ NO: 66) is provided. Inanother embodiment any one of ARC 330 (SEQ ID NO: 2) and ARC188-189,ARC250, ARC296-297, ARC331-334, ARC411-440, ARC457-459, ARC473,ARC522-525, ARC532, ARC543-544, ARC550-554, ARC657-658, ARC672, ARC706,ARC1537, ARC1730, (SEQ ID NOS: 6 to SEQ NO: 66) for use in thepreparation of a pharmaceutical composition is provided. In this aspect,the invention provides a pharmaceutical composition comprising atherapeutically effective amount of an aptamer that inhibits C5complement protein cleavage in vivo or a salt thereof and apharmaceutically acceptable carrier or diluent.

In a particular embodiment, an aptamer comprising a nucleotide sequenceaccording to SEQ ID NO: 1 is provided. In a particular embodiment, anaptamer comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64 to SEQ IDNO: 66 is provided. In some embodiments, where the aptamer comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 61,SEQ ID NO: 62, and SEQ ID NO: 64 to SEQ ID NO: 66, the aptamer comprisessubstantially the same binding affinity for C5 complement protein as anaptamer consisting of the sequence according to SEQ ID NO: 4 but lackinga PEG moiety.

In some embodiments wherein the aptamer comprises a nucleotide sequenceselected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, andSEQ ID NO: 64 to SEQ ID NO: 66, the aptamer comprises a half life,preferably the terminal half life in a two compartment model asdetermined in Example 5E below, of at least 15, preferably at least 30hours in primate. In some embodiments wherein the aptamer comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 61,SEQ ID NO: 62, and SEQ ID NO: 64 to SEQ ID NO: 66, the aptamer comprisesa half life, preferably the terminal half life in a two compartmentmodel, of at least 1 and a half, preferably at least seven hours in rat.

In some embodiments of this aspect of the invention, wherein the aptamercomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64 to SEQ ID NO: 66, theaptamer is synthesized with a 5′ linker as follows: H₂N

5′ Aptamer 3′, wherein

denotes the linker. In some embodiments the linker is an alkyl linker asfollows: H2N—(CH2)_(n)-5′ Aptamer 3′ wherein n=2 to 18, preferablyn=2-12, more preferably n=3 to 6, more preferably n=6, and wherein

-   Aptamer=fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfJfU    AfCfCfUmGfCmG-3T (SEQ ID NO: 4)    wherein fC and fU=2′-fluoro nucleotides, and mG and mA=2′-OMe    nucleotides and all other nucleotides are 2′-OH and where 3T    indicates an inverted deoxy thymidine. The resulting amine-modified    aptamer may be conjugated to a PEG moiety selected from the group    consisting of a 10 kDa PEG, 20 kDa PEG, 30 kDa PEG and 40 kDa linear    PEG. In some embodiments, a pharmaceutical composition comprising a    therapeutically effective amount of an aptamer comprising a    nucleotide sequence selected from the group consisting of: SEQ ID    NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO: 66, particularly    from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ    ID NO: 64 to SEQ ID NO: 66 or a salt thereof is provided. The    pharmaceutical composition of the invention may comprise a    pharmaceutically acceptable carrier or diluent. In this aspect of    the invention a pharmaceutical composition for use in the treatment,    prevention or amelioration of disease in vivo, comprising an aptamer    which comprises a nucleotide sequence selected from the group    consisting of: SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO: 66,    particularly from the group consisting of SEQ ID NO: 61, SEQ ID NO:    62, and SEQ ID NO: 64 to SEQ ID NO: 66 is provided.

In another embodiment, a method of treating, preventing or amelioratinga disease mediated by C5 complement protein is provided, comprisingadministering a pharmaceutical composition comprising an aptamer or asalt thereof, where the aptamer comprises a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO:66, particularly from the group consisting of SEQ ID NO: 61, SEQ ID NO:62, and SEQ ID NO: 64 to SEQ ID NO: 66 to a vertebrate. In someembodiments of this aspect of the invention, the method comprisesadministering the pharmaceutical composition of the invention to amammal, preferably a human.

In some embodiments, the C5 complement protein, C5a and/orC5b-9-mediated disease to be treated is acute ischemic diseases(myocardial infarction, stroke, ischemic/reperfusion injury); acuteinflammatory diseases (infectious disease, septicemia, shock,acute/hyperacute transplant rejection); chronic inflammatory and/orimmune-mediated diseases (allergy, asthma, rheumatoid arthritis, andother rheumatological diseases, multiple sclerosis and otherneurological diseases, psoriasis and other dermatological diseases,myasthenia gravis, systemic lupus erythematosus (SLE), subacute/chronictransplant rejection, glomerulonephritis and other renal diseases). Insome embodiments, the C5 complement protein, C5a and/or C5b-9 mediateddiseases to be treated include complement activation associated withdialysis or circumstances in which blood is passed over and/or throughsynthetic tubing and/or foreign material. In some embodiments, the C5complement protein C5a and/or C5b-9-mediated disease to be treated isselected from the group consisting of myocardial injury relating to CABGsurgery, myocardial injury relating to balloon angioplasty andmyocardial injury relating to restenosis. In some embodiments, C5complement protein, C5a and/or C5b-9-mediated disorder to be treated isselected from the group consisting of: myocardial injury relating toCABG surgery, myocardial injury relating to balloon angioplasty,myocardial injury relating to restenosis, complement protein mediatedcomplications relating to CABG surgery, complement protein mediatedcomplications relating to percutaneous coronary intervention,paroxysomal nocturnal hemoglobinuria, acute transplant rejection,hyperacute transplant rejection, subacute transplant rejection, andchronic transplant rejection. In some embodiments the C5 complementprotein C5a and/or C5b-9-mediated disease to be treated is complicationsrelating to CABG surgery. In a particular embodiment, the disease to betreated is myocardial injury relating to CABG surgery.

In some embodiments, the method of the invention includes administeringthe pharmaceutical composition comprising an aptamer having a nucleotidesequence selected from the group consisting of: SEQ ID NO: 2 and SEQ IDNO: 6 to SEQ NO: 66, particularly from the group consisting of SEQ IDNO: 61, SEQ ID NO: 62, and SEQ ID NO: 64 to SEQ ID NO: 66, to a patientto achieve an aptamer plasma concentration that is about 0.5 to about 10times that of the endogenous C5 complement protein. In some embodiments,the pharmaceutical aptamer compositions are administered to achieve anaptamer plasma concentration that is about 0.75 to about 5 times, 0.75to about 3 times, and 1.5 to about 2 times that of the endogenous C5complement protein while in other embodiments the aptamer composition isadministered to achieve a concentration equivalent to that of theendogenous complement protein. In some embodiments, the pharmaceuticalcomposition of the invention administered to achieve an aptamer plasmaconcentration of about 5 μM, about 4 μM, about 3 μM, about 2 μM, about1.5 μM, about 1 μM or of about 500 nM.

Any combination of route, duration, and rate of administration may beused that is sufficient to achieve the aptamer plasma concentrations ofthe invention. In some embodiments the pharmaceutical composition isadministered intravenously. In some embodiments, the pharmaceuticalcomposition is administered as a bolus and/or via continuous infusion.

In particular embodiments of treating, preventing and/or amelioratingcomplications related to CABG surgery, particularly myocardial injuryrelated to CABG surgery, the method of the invention comprisesadministering the pharmaceutical composition prior to surgery andcontinuing administration at least 24 hours, in some embodiments about48 hours or in some embodiments about 72 hours. In a particularembodiment of this aspect of the invention, the desired aptamer plasmaconcentration, e.g., two times the endogenous complement proteinconcentration in some embodiments, is achieved by administration of anintravenous bolus to the patient to be treated in advance of,simultaneously with, or after intravenous infusion of a lower dose ofaptamer. In still other embodiments of this aspect of the invention,where the aptamer/conjugate comprises a sufficiently long half life, theaptamer pharmaceutical composition may be administered once or twicedaily as an intravenous bolus dose.

In another aspect of the invention diagnostic methods are provided. Inone embodiment, the diagnostic method comprises contacting a compositionsuspected of comprising C5 complement protein or a variant thereof withan aptamer comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO 66, particularlyfrom the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ IDNO: 64 to SEQ ID NO: 66, and detecting the presence or absence of C5complement protein or a variant thereof. In some embodiments thecomplement protein or variant is vertebrate, particularly mammalian, andmore particularly human. The present invention provides an aptamercomposition having an aptamer comprising a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO 66for use as an in vitro or in vivo diagnostic. In the present invention,an aptamer comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO: 2 and SEQ ID NO: 6 to SEQ NO 66 for use in thepreparation of a pharmaceutical composition is provided.

In another aspect of the invention, an aptamer comprising a nucleotidesequence that is 80% identical to any one of the sequences selected fromthe group consisting of SEQ ID NOS: 75 to 81, SEQ ID NO: 83, and SEQ IDNOS: 88 to 98 is provided. In some embodiments, an aptamer comprising anucleotide sequence that is 80% identical to the unique region of anyone of the sequences selected from the group consisting of SEQ ID NOS:75 to 81 and SEQ ID NOS: 88 to 98 is provided. In another embodiment anaptamer comprising a nucleotide sequence that is 90% identical to anyone of the sequences selected from the group consisting of SEQ ID NOS:75 to 81, SEQ ID NO: 83, and SEQ ID NOS: 88 to 98 is provided. In aparticular embodiment, an aptamer comprising a nucleotide sequence thatis 90% identical to the unique region of any one of the sequencesselected from the group consisting of SEQ ID NOS: 75 to 81 and SEQ IDNOS: 88 to 98 is provided. In yet another embodiment, an aptamercomprising a nucleotide sequence of 40 contiguous nucleotides identicalto 40 contiguous nucleotides included in any one of the sequencesselected from the group consisting of SEQ ID NOS: 75 to 81 and SEQ IDNOS: 88 to 98 is provided. In another embodiment, an aptamer comprisinga nucleotide sequence of 30 contiguous nucleotides identical to 30contiguous nucleotides included in any one of the sequences selectedfrom the group consisting of SEQ ID NOS: 75 to 81, SEQ ID NO: 83 and SEQID NOS: 88 to 98 is provided. In yet another embodiment, an aptamer thatbinds specifically to C5 complement protein comprising a nucleotidesequence of 10 contiguous nucleotides identical to 10 contiguousnucleotides included in any one of the sequences selected from the groupconsisting of SEQ ID NOS: 75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to98 is provided. In a preferred embodiment an aptamer comprising anucleotide sequence according to any one of the nucleotide sequencesselected from the group consisting of: SEQ ID NOS: 75 to 81, SEQ ID NO:83 and SEQ ID NOS: 88 to 98, is provided.

In some embodiments, the aptamers of this aspect of the inventiondescribed immediately above may further comprise a chemical modificationselected from the group consisting: of a chemical substitution at asugar position; a chemical substitution at a phosphate position; and achemical substitution at a base position of the nucleic acid sequence.In some embodiments the modification is selected from the groupconsisting of: incorporation of a modified nucleotide; 3′ capping,conjugation to a high molecular weight, non-immunogenic compound;conjugation to a lipophilic compound; and modification of the phosphateback bone.

In preferred embodiments of this aspect of the invention, the aptamermodulates a function of a C5 complement protein or a variant thereof. Inparticularly preferred embodiments, the aptamer inhibits a function ofC5 complement protein or a variant thereof, preferably in vivo, morepreferably in vivo in humans. In one embodiment of this aspect of theinvention, the function modulated, preferably inhibited, by the aptameris C5 complement protein cleavage.

In some embodiments of another aspect, the invention provides apharmaceutical composition comprising a therapeutically effective amountof an aptamer that blocks C5 complement protein cleavage in vivo or asalt thereof and a pharmaceutically acceptable carrier or diluent.

In some embodiments, a pharmaceutical composition comprising atherapeutically effective amount of an aptamer comprising a nucleotidesequence 80% identical to, preferably 90% identical to a nucleotidesequence selected from the group consisting of SEQ ID NOS: 75 to 81, SEQID NO: 83 and SEQ ID NOS: 88 to 98 or a salt thereof is provided. Insome embodiments, a pharmaceutical composition comprising atherapeutically effective amount of an aptamer comprising a nucleotidesequence 80% identical to, preferably 90% identical to the unique regionof a nucleotide sequence selected from the group consisting of SEQ IDNOS: 75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98 or a salt thereofis provided. In other embodiments, a pharmaceutical compositioncomprising a therapeutically effective amount of an aptamer having 40,30 or 10 contiguous nucleotides identical to 40, 30 or 10 nucleotides,respectively, to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to98 is provided. The pharmaceutical composition of the invention maycomprise a pharmaceutically acceptable carrier or diluent. In thisaspect of the invention a pharmaceutical composition is provided for usein the treatment, prevention or amelioration of disease in vivo, wherethe pharmaceutical composition comprises an aptamer having a nucleotidesequence selected from the group consisting of: SEQ ID NOS: 3 to 4, SEQID NOS: 75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98 or a saltthereof. In this aspect, an aptamer having a nucleotide sequenceselected from the group consisting of: SEQ ID NOS: 3 to 4, SEQ ID NOS:75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98 for use in thepreparation of a pharmaceutical composition is provided. In this aspect,the invention provides a pharmaceutical composition comprising atherapeutically effective amount of an aptamer that inhibits C5complement protein cleavage in vivo or a salt thereof and apharmaceutically acceptable carrier or diluent.

In some embodiments, the C5 complement protein, C5a and/orC5b-9-mediated disease to be treated is acute ischemic diseases(myocardial infarction, stroke, ischemic/reperfusion injury); acuteinflammatory diseases (infectious disease, septicemia, shock,acute/hyperacute transplant rejection); chronic inflammatory and/orimmune-mediated diseases (allergy, asthma, rheumatoid arthritis, andother rheumatological diseases, multiple sclerosis and otherneurological diseases, psoriasis and other dermatological diseases,myasthenia gravis, systemic lupus erythematosus (SLE), subacute/chronictransplant rejection, glomerulonephritis and other renal diseases). Insome embodiments, the C5 complement protein, C5a and/or C5b-9 mediateddiseases to be treated include complement activation associated withdialysis or circumstances in which blood is passed over and/or throughsynthetic tubing and/or foreign material. In some embodiments, the C5complement protein C5a and/or C5b-9-mediated disease to be treated isselected from the group consisting of myocardial injury relating to CABGsurgery, myocardial injury relating to balloon angioplasty andmyocardial injury relating to restenosis. In some embodiments, C5complement protein, C5a and/or C5b-9-mediated disorder to be treated isselected from the group consisting of: myocardial injury relating toCABG surgery, myocardial injury relating to balloon angioplasty,myocardial injury relating to restenosis, complement protein mediatedcomplications relating to CABG surgery, complement protein mediatedcomplications relating to percutaneous coronary intervention, paroxysmalnocturnal hemoglobinuria, acute transplant rejection, hyperacutetransplant rejection, subacute transplant rejection, and chronictransplant rejection. In some embodiments the C5 complement protein C5aand/or C5b-9-mediated disease to be treated is complications relating toCABG surgery. In a particular embodiment, the disease to be treated ismyocardial injury relating to CABG surgery.

In some embodiments, the method of the invention includes administeringthe pharmaceutical composition comprising an aptamer having a nucleotidesequence selected from the group consisting of: SEQ ID NOS: 3 to 4, SEQID NOS: 75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98, to a patientto achieve an aptamer plasma concentration that is about 0.5 to about 10times that of the endogenous C5 complement protein. In some embodiments,the pharmaceutical aptamer compositions are administered to achieve anaptamer plasma concentration that is about 0.75 to about 5 times, 0.75to about 3 times, and 1.5 to about 2 times that of the endogenous C5complement protein while in other embodiments the aptamer composition isadministered to achieve a concentration equivalent to that of theendogenous complement protein. In some embodiments, the pharmaceuticalcomposition of the invention administered to achieve an aptamer plasmaconcentration of about 5 μM, about 4 μM, about 3 μM, about 2 μM, about1.5 μM, about 1 μM or of about 500 nM.

Any combination of route, duration, and rate of administration may beused that is sufficient to achieve the aptamer plasma concentrations ofthe invention. In some embodiments the pharmaceutical composition isadministered intravenously. In some embodiments, the pharmaceuticalcomposition is administered as a bolus and/or via continuous infusion.

In particular embodiments of treating, preventing and/or amelioratingcomplications related to CABG surgery, particularly myocardial injuryrelated to CABG surgery, the method of the invention comprisesadministering the pharmaceutical composition prior to surgery andcontinuing administration at least 24 hours, in some embodiments about48 hours or in some embodiments about 72 hours. In a particularembodiment of this aspect of the invention, the desired aptamer plasmaconcentration, e.g., two times the endogenous complement proteinconcentration in some embodiments, is achieved by administration of anintravenous bolus to the patient to be treated in advance of,simultaneously with or after intravenous infusion of a lower dose ofaptamer. In still other embodiments of this aspect of the invention,where the aptamer/conjugate comprises a sufficiently long half life, theaptamer pharmaceutical composition may be administered once or twicedaily as an intravenous bolus dose.

In another embodiment, a diagnostic method is provided, the methodcomprising contacting a composition suspected of comprising C5complement protein or a variant thereof with an aptamer comprising anucleotide sequence selected from the group consisting of: SEQ ID NOS:75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98 and detecting thepresence or absence of C5 complement protein or a variant thereof. Insome embodiments the complement protein or variant is vertebrate,particularly mammalian, and more particularly human. The presentinvention provides an aptamer composition having an aptamer comprising anucleotide sequence selected from the group consisting of: SEQ ID NOS:75 to 81, SEQ ID NO: 83 and SEQ ID NOS: 88 to 98 for use as an in vitroor in vivo diagnostic.

In some embodiments, an aptamer comprising a nucleotide sequenceconsisting essentially of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 68 and 69 is provided. In some embodiments, anaptamer comprising a nucleotide sequence consisting of a nucleotidesequence selected from the group consisting of SEQ ID NO: 68 and 69 isprovided. In some embodiments of this aspect of the invention, theaptamers may be used in a diagnostic method.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present Specificationwill control.

The SELEX™ Method

A suitable method for generating an aptamer is with the process entitled“Systematic Evolution of Ligands by Exponential Enrichment” (“SELEX™”)generally depicted in FIG. 2. The SELEX™ process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in, e.g., U.S. patent applicationSer. No. 07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163(see also WO 91/19813) entitled “Nucleic Acid Ligands”. EachSELEX™-identified nucleic acid ligand, i.e., each aptamer, is a specificligand of a given target compound or molecule. The SELEX™ process isbased on the unique insight that nucleic acids have sufficient capacityfor forming a variety of two- and three-dimensional structures andsufficient chemical versatility available within their monomers to actas ligands (i.e., form specific binding pairs) with virtually anychemical compound, whether monomeric or polymeric. Molecules of any sizeor composition can serve as targets.

SELEX™ relies as a starting point upon a large library or pool of singlestranded oligonucleotides comprising randomized sequences. Theoligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNAhybrids. In some examples, the pool comprises 100% random or partiallyrandom oligonucleotides. In other examples, the pool comprises random orpartially random oligonucleotides containing at least one fixed and/orconserved sequence incorporated within randomized sequence. In otherexamples, the pool comprises random or partially random oligonucleotidescontaining at least one fixed and/or conserved sequence at its 5′ and/or3′ end which may comprise a sequence shared by all the molecules of theoligonucleotide pool. Fixed sequences are sequences such ashybridization sites for PCR primers, promoter sequences for RNApolymerases (e.g., T3, T4, T7, and SP6), restriction sites, orhomopolymeric sequences, such as poly A or poly T tracts, catalyticcores, sites for selective binding to affinity columns, and othersequences to facilitate cloning and/or sequencing of an oligonucleotideof interest. Conserved sequences are sequences, other than thepreviously described fixed sequences, shared by a number of aptamersthat bind to the same target.

The oligonucleotides of the pool preferably include a randomizedsequence portion as well as fixed sequences necessary for efficientamplification. Typically the oligonucleotides of the starting poolcontain fixed 5′ and 3′ terminal sequences which flank an internalregion of 30-50 random nucleotides. The randomized nucleotides can beproduced in a number of ways including chemical synthesis and sizeselection from randomly cleaved cellular nucleic acids. Sequencevariation in test nucleic acids can also be introduced or increased bymutagenesis before or during the selection/amplification iterations.

The random sequence portion of the oligonucleotide can be of any lengthand can comprise ribonucleotides and/or deoxyribonucleotides and caninclude modified or non-natural nucleotides or nucleotide analogs. See,e.g., U.S. Pat. Nos. 5,958,691; 5,660,985; 5,958,691; 5,698,687;5,817,635; 5,672,695, and PCT Publication WO 92/07065. Randomoligonucleotides can be synthesized from phosphodiester-linkednucleotides using solid phase oligonucleotide synthesis techniques wellknown in the art. See, e.g., Froehler et al., Nucl. Acid Res.14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578 (1986).Random oligonucleotides can also be synthesized using solution phasemethods such as triester synthesis methods. See, e.g., Sood et al.,Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449(1978). Typical syntheses carried out on automated DNA synthesisequipment yield 10¹⁴-10¹⁶ individual molecules, a number sufficient formost SELEX™ experiments. Sufficiently large regions of random sequencein the sequence design increases the likelihood that each synthesizedmolecule is likely to represent a unique sequence.

The starting library of oligonucleotides may be generated by automatedchemical synthesis on a DNA synthesizer. To synthesize randomizedsequences, mixtures of all four nucleotides are added at each nucleotideaddition step during the synthesis process, allowing for randomincorporation of nucleotides. As stated above, in one embodiment, randomoligonucleotides comprise entirely random sequences; however, in otherembodiments, random oligonucleotides can comprise stretches of nonrandomor partially random sequences. Partially random sequences can be createdby adding the four nucleotides in different molar ratios at eachaddition step.

The starting library of oligonucleotides may be either RNA or DNA. Inthose instances where an RNA library is to be used as the startinglibrary it is typically generated by transcribing a DNA library in vitrousing T7 RNA polymerase or modified T7 RNA polymerases and purified. TheRNA or DNA library is then mixed with the target under conditionsfavorable for binding and subjected to step-wise iterations of binding,partitioning and amplification, using the same general selection scheme,to achieve virtually any desired criterion of binding affinity andselectivity. More specifically, starting with a mixture containing thestarting pool of nucleic acids, the SELEX™ method includes steps of: (a)contacting the mixture with the target under conditions favorable forbinding; (b) partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules; (c) dissociating thenucleic acid-target complexes; (d) amplifying the nucleic acidsdissociated from the nucleic acid-target complexes to yield aligand-enriched mixture of nucleic acids; and (e) reiterating the stepsof binding, partitioning, dissociating and amplifying through as manycycles as desired to yield highly specific, high affinity nucleic acidligands to the target molecule. In those instances where RNA aptamersare being selected, the SELEX™ method further comprises the steps of:(i) reverse transcribing the nucleic acids dissociated from the nucleicacid-target complexes before amplification in step (d); and (ii)transcribing the amplified nucleic acids from step (d) before restartingthe process.

Within a nucleic acid mixture containing a large number of possiblesequences and structures, there is a wide range of binding affinitiesfor a given target. A nucleic acid mixture comprising, for example, a 20nucleotide randomized segment can have 4²⁰ candidate possibilities.Those which have the higher affinity constants for the target are mostlikely to bind to the target. After partitioning, dissociation andamplification, a second nucleic acid mixture is generated, enriched forthe higher binding affinity candidates. Additional rounds of selectionprogressively favor the best ligands until the resulting nucleic acidmixture is predominantly composed of only one or a few sequences. Thesecan then be cloned, sequenced and individually tested for bindingaffinity as pure ligands or aptamers.

Cycles of selection and amplification are repeated until a desired goalis achieved. In the most general case, selection/amplification iscontinued until no significant improvement in binding strength isachieved on repetition of the cycle. The method is typically used tosample approximately 10¹⁴ different nucleic acid species but may be usedto sample as many as about 10¹⁸ different nucleic acid species.Generally, nucleic acid aptamer molecules are selected in a 5 to 20cycle procedure. In one embodiment, heterogeneity is introduced only inthe initial selection stages and does not occur throughout thereplicating process.

In one embodiment of SELEX™, the selection process is so efficient atisolating those nucleic acid ligands that bind most strongly to theselected target, that only one cycle of selection and amplification isrequired. Such an efficient selection may occur, for example, in achromatographic-type process wherein the ability of nucleic acids toassociate with targets bound on a column operates in such a manner thatthe column is sufficiently able to allow separation and isolation of thehighest affinity nucleic acid ligands.

In many cases, it is not necessarily desirable to perform the iterativesteps of SELEX™ until a single nucleic acid ligand is identified. Thetarget-specific nucleic acid ligand solution may include a family ofnucleic acid structures or motifs that have a number of conservedsequences and a number of sequences which can be substituted or addedwithout significantly affecting the affinity of the nucleic acid ligandsto the target. By terminating the SELEX™ process prior to completion, itis possible to determine the sequence of a number of members of thenucleic acid ligand solution family.

A variety of nucleic acid primary, secondary and tertiary structures areknown to exist. The structures or motifs that have been shown mostcommonly to be involved in non-Watson-Crick type interactions arereferred to as hairpin loops, symmetric and asymmetric bulges,pseudoknots and myriad combinations of the same. Almost all known casesof such motifs suggest that they can be formed in a nucleic acidsequence of no more than 30 nucleotides. For this reason, it is oftenpreferred that SELEX™ procedures with contiguous randomized segments beinitiated with nucleic acid sequences containing a randomized segment ofbetween about 20 to about 50 nucleotides and in some embodiments ofabout 30 to about 40 nucleotides. In one example, the5′-fixed:random:3′-fixed sequence comprises a random sequence of about30 to about 50 nucleotides.

The core SELEX™ method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796 describes the use ofSELEX™ in conjunction with gel electrophoresis to select nucleic acidmolecules with specific structural characteristics, such as bent DNA.U.S. Pat. No. 5,763,177 describes SELEX™ based methods for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. Pat. Nos. 5,567,588 and 5,861,254 describe SELEX™ based methodswhich achieve highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule. U.S. Pat. No.5,496,938 describes methods for obtaining improved nucleic acid ligandsafter the SELEX™ process has been performed. U.S. Pat. No. 5,705,337describes methods for covalently linking a ligand to its target.

SELEX™ can also be used to obtain nucleic acid ligands that bind to morethan one site on the target molecule, and to obtain nucleic acid ligandsthat include non-nucleic acid species that bind to specific sites on thetarget. SELEX™ provides means for isolating and identifying nucleic acidligands which bind to any envisionable target, including large and smallbiomolecules such as nucleic acid-binding proteins and proteins notknown to bind nucleic acids as part of their biological function as wellas cofactors and other small molecules. For example, U.S. Pat. No.5,580,737 discloses nucleic acid sequences identified through SELEX™which are capable of binding with high affinity to caffeine and theclosely related analog, theophylline.

Counter-SELEX™ is a method for improving the specificity of nucleic acidligands to a target molecule by eliminating nucleic acid ligandsequences with cross-reactivity to one or more non-target molecules.Counter-SELEX™ is comprised of the steps of: (a) preparing a candidatemixture of nucleic acids; (b) contacting the candidate mixture with thetarget, wherein nucleic acids having an increased affinity to the targetrelative to the candidate mixture may be partitioned from the remainderof the candidate mixture; (c) partitioning the increased affinitynucleic acids from the remainder of the candidate mixture; (d)dissociating the increased affinity nucleic acids from the target; e)contacting the increased affinity nucleic acids with one or morenon-target molecules such that nucleic acid ligands with specificaffinity for the non-target molecule(s) are removed; and f) amplifyingthe nucleic acids with specific affinity only to the target molecule toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity and specificity for binding to thetarget molecule. As described above for SELEX™, cycles of selection andamplification are repeated as necessary until a desired goal isachieved.

One potential problem encountered in the use of nucleic acids astherapeutics and vaccines is that oligonucleotides in theirphosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. The SELEX™ methodthus encompasses the identification of high-affinity nucleic acidligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX™-identified nucleic acid ligands containingmodified nucleotides are described, e.g., in U.S. Pat. No. 5,660,985,which describes oligonucleotides containing nucleotide derivativeschemically modified at the 2′ position of ribose, 5 position ofpyrimidines, and 8 position of purines, U.S. Pat. No. 5,756,703 whichdescribes oligonucleotides containing various 2′-modified pyrimidines,and U.S. Pat. No. 5,580,737 which describes highly specific nucleic acidligands containing one or more nucleotides modified with 2′-amino(2′-NH2), 2′-fluoro (2′-F), and/or 2′-OMe (2′-OMe) substituents.

Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Modifications to generate oligonucleotide populationswhich are resistant to nucleases can also include one or more substituteinternucleotide linkages, altered sugars, altered bases, or combinationsthereof. Such modifications include, but are not limited to, 2′-positionsugar modifications, 5-position pyrimidine modifications, 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, phosphorothioate or alkyl phosphate modifications,methylations, and unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine. Modifications can also include 3′ and 5′modifications such as capping.

In one embodiment, oligonucleotides are provided in which the P(O)Ogroup is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR2(“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”) or 3′-amine(—NH—CH2-CH2-), wherein each R or R′ is independently H or substitutedor unsubstituted alkyl. Linkage groups can be attached to adjacentnucleotides through an —O—, —N—, or —S— linkage. Not all linkages in theoligonucleotide are required to be identical. As used herein, the termphosphorothioate encompasses one or more non-bridging oxygen atoms in aphosphodiester bond replaced by one or more sulfur atoms.

In further embodiments, the oligonucleotides comprise modified sugargroups, for example, one or more of the hydroxyl groups is replaced withhalogen, aliphatic groups, or functionalized as ethers or amines. In oneembodiment, the 2′-position of the furanose residue is substituted byany of an OMe, O-alkyl, O-alklyl, S-alkyl, S-allyl, or halo group.Methods of synthesis of 2′-modified sugars are described, e.g., inSproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, et al., Nucl.Acid Res. 19:2629-2635 (1991); and Hobbs, et al., Biochemistry12:5138-5145 (1973). Other modifications are known to one of ordinaryskill in the art. Such modifications may be pre-SELEX™ processmodifications or post-SELEX™ process modifications (modification ofpreviously identified unmodified ligands) or may be made byincorporation into the SELEX process.

Pre-SELEX process modifications or those made by incorporation into theSELEX process yield nucleic acid ligands with both specificity for theirSELEX™ target and improved stability, e.g., in vivo stability.Post-SELEX™ process modifications made to nucleic acid ligands mayresult in improved stability, e.g., in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

The SELEX™ method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. Nos. 5,637,459 and 5,683,867. The SELEX™method further encompasses combining selected nucleic acid ligands withlipophilic or non-immunogenic high molecular weight compounds in adiagnostic or therapeutic complex, as described, e.g., in U.S. Pat. Nos.6,011,020, 6,051,698, and PCT Publication No. WO 98/18480. These patentsand applications teach the combination of a broad array of shapes andother properties, with the efficient amplification and replicationproperties of oligonucleotides, and with the desirable properties ofother molecules.

The identification of nucleic acid ligands to small, flexible peptidesvia the SELEX™ method has also been explored. Small peptides haveflexible structures and usually exist in solution in an equilibrium ofmultiple conformers, and thus it was initially thought that bindingaffinities may be limited by the conformational entropy lost uponbinding a flexible peptide. However, the feasibility of identifyingnucleic acid ligands to small peptides in solution was demonstrated inU.S. Pat. No. 5,648,214. In this patent, high affinity RNA nucleic acidligands to substance P, an 11 amino acid peptide, were identified.

The aptamers with specificity and binding affinity to the target(s) ofthe present invention are typically selected by the SELEX™ process asdescribed herein. As part of the SELEX™ process, the sequences selectedto bind to the target are then optionally minimized to determine theminimal sequence having the desired binding affinity. The selectedsequences and/or the minimized sequences are optionally optimized byperforming random or directed mutagenesis of the sequence to increasebinding affinity or alternatively to determine which positions in thesequence are essential for binding activity.

Additionally, selections can be performed with sequences incorporatingmodified nucleotides to stabilize the aptamer molecules againstdegradation in vivo.

2′Modified SELEX™

In order for an aptamer to be suitable for use as a therapeutic, it ispreferably inexpensive to synthesize, safe and stable in vivo. Wild-typeRNA and DNA aptamers are typically not stable in vivo because of theirsusceptibility to degradation by nucleases. Resistance to nucleasedegradation can be greatly increased by the incorporation of modifyinggroups at the 2′-position.

Fluoro and amino groups have been successfully incorporated intooligonucleotide libraries from which aptamers have been subsequentlyselected. However, these modifications greatly increase the cost ofsynthesis of the resultant aptamer, and may introduce safety concerns insome cases because of the possibility that the modified nucleotidescould be recycled into host DNA by degradation of the modifiedoligonucleotides and subsequent use of the nucleotides as substrates forDNA synthesis.

Aptamers that contain 2′-OMe (“2′-OMe”) nucleotides, as provided in someembodiments herein, overcome many of these drawbacks. Oligonucleotidescontaining 2′-OMe nucleotides are nuclease-resistant and inexpensive tosynthesize. Although 2′-OMe nucleotides are ubiquitous in biologicalsystems, natural polymerases do not accept 2′-O-methyl NTPs assubstrates under physiological conditions, thus there are no safetyconcerns over the recycling of 2′-OMe nucleotides into host DNA. TheSELEX™ method used to generate 2′-modified aptamers is described, e.g.,in U.S. Provisional Patent Application Ser. No. 60/430,761, filed Dec.3, 2002, U.S. Provisional Patent Application Ser. No. 60/487,474, filedJul. 15, 2003, U.S. Provisional Patent Application Ser. No. 60/517,039,filed Nov. 4, 2003, U.S. patent application Ser. No. 10/729,581, filedDec. 3, 2003, and U.S. patent application Ser. No. 10/873,856, filedJun. 21, 2004, entitled “Method for in vitro Selection of 2′-OMeSubstituted Nucleic Acids”, each of which is herein incorporated byreference in its entirety.

The present invention includes aptamers that bind to and modulate thefunction of complement protein C5 which contain modified nucleotides(e.g., nucleotides which have a modification at the 2′position) to makethe oligonucleotide more stable than the unmodified oligonucleotide toenzymatic and chemical degradation as well as thermal and physicaldegradation. Although there are several examples of 2′-OMe containingaptamers in the literature (see, e.g., Green et al., Current Biology 2,683-695, 1995) these were generated by the in vitro selection oflibraries of modified transcripts in which the C and U residues were2′-fluoro (2′-F) substituted and the A and G residues were 2′-OH. Oncefunctional sequences were identified then each A and G residue wastested for tolerance to 2′-OMe substitution, and the aptamer wasre-synthesized having all A and G residues which tolerated 2′-OMesubstitution as 2′-OMe residues. Most of the A and G residues ofaptamers generated in this two-step fashion tolerate substitution with2′-OMe residues, although, on average, approximately 20% do not.Consequently, aptamers generated using this method tend to contain fromtwo to four 2′-OH residues, and stability and cost of synthesis arecompromised as a result. By incorporating modified nucleotides into thetranscription reaction which generate stabilized oligonucleotides usedin oligonucleotide pools from which aptamers are selected and enrichedby SELEX™ (and/or any of its variations and improvements, includingthose described herein), the methods of the present invention eliminatethe need for stabilizing the selected aptamer oligonucleotides (e.g., byresynthesizing the aptamer oligonucleotides with modified nucleotides).

In one embodiment, the present invention provides aptamers comprisingcombinations of 2′-OH, 2′-F, 2′-deoxy, and 2′-OMe modifications of theATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, thepresent invention provides aptamers comprising combinations of 2′-OH,2′-F, 2′-deoxy, 2′-OMe, 2′-NH2, and 2′-methoxyethyl modifications of theATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, thepresent invention provides aptamers comprising 5⁶ combinations of 2′-OH,2′-F, 2′-deoxy, 2′-OMe, 2′-NH2, and 2′-inethoxyethyl modifications ofthe ATP, GTP, CTP, TTP, and UTP nucleotides.

2′ modified aptamers of the invention are created using modifiedpolymerases, e.g., a modified T7 polymerase, having a rate ofincorporation of modified nucleotides having bulky substituents at thefuranose 2′ position that is higher than that of wild-type polymerases.For example, a single mutant T7 polymerase (Y639F) in which the tyrosineresidue at position 639 has been changed to phenylalanine readilyutilizes 2′deoxy, 2′amino-, and 2′fluoro-nucleotide triphosphates (NTPs)as substrates and has been widely used to synthesize modified RNAs for avariety of applications. However, this mutant T7 polymerase reportedlycan not readily utilize (i.e., incorporate) NTPs with bulky2′-substituents such as 2′-OMe or 2′-azido (2′-N3) substituents. Forincorporation of bulky 2′ substituents, a double T7 polymerase mutant(Y639F/H784A) having the histidine at position 784 changed to an alanineresidue in addition to the Y639F mutation has been described and hasbeen used in limited circumstances to incorporate modified pyrimidineNTPs. See Padilla, R. and Sousa, R., Nucleic Acids Res., 2002, 30(24):138. A single mutant T7 polymerase (H784A) having the histidine atposition 784 changed to an alanine residue has also been described.Padilla at al., Nucleic Acids Research, 2002, 30: 138. In both theY639F/H784A double mutant and H784A single mutant T7 polymerases, thechange to a smaller amino acid residue such as alanine allows for theincorporation of bulkier nucleotide substrates, e.g., 2′-O methylsubstituted nucleotides.

Generally, it has been found that under the conditions disclosed herein,the Y693F single mutant can be used for the incorporation of all 2′-OMesubstituted NTPs except GTP and the Y639F/F1784A double mutant can beused for the incorporation of all 2′-OMe substituted NTPs including GTP.It is expected that the H784A single mutant possesses properties similarto the Y639F and the Y639F/H784A mutants when used under the conditionsdisclosed herein.

2′-modified oligonucleotides may be synthesized entirely of modifiednucleotides, or with a subset of modified nucleotides. The modificationscan be the same or different. All nucleotides may be modified, and allmay contain the same modification. All nucleotides may be modified, butcontain different modifications, e.g., all nucleotides containing thesame base may have one type of modification, while nucleotidescontaining other bases may have different types of modification. Allpurine nucleotides may have one type of modification (or areunmodified), while all pyrimidine nucleotides have another, differenttype of modification (or are unmodified). In this way, transcripts, orpools of transcripts are generated using any combination ofmodifications, including for example, ribonucleotides (2′-OH),deoxyribonucleotides (2′-deoxy), 2′-F, and 2′-OMe nucleotides. Atranscription mixture containing 2′-OMe C and U and 2′-OH A and G isreferred to as a “rRmY” mixture and aptamers selected therefrom arereferred to as “rRmY” aptamers. A transcription mixture containing deoxyA and G and 2′-OMe U and C is referred to as a “dRmY” mixture and.aptamers selected therefrom are referred to as “dRmY” aptamers. Atranscription mixture containing 2′-OMe A, C, and U, and 2′-OH G isreferred to as a “rGmH” mixture and aptamers selected therefrom arereferred to as “rGmH” aptamers. A transcription mixture alternatelycontaining 2′-OMe A, C, U and G and 2′-OMe A, U and C and 2′-F G isreferred to as a “alternating mixture’ and aptamers selected therefromare referred to as “alternating mixture” aptamers. A transcriptionmixture containing 2′-OMe A, U, C, and G, where up to 10% of the G's areribonucleotides is referred to as a “r/mGmH” mixture and aptamersselected therefrom are referred to as “r/mGmH” aptamers. A transcriptionmixture containing 2′-OMe A, U, and C, and 2′-F G is referred to as a“fGmH” mixture and aptamers selected therefrom are referred to as “fGmH”aptamers. A transcription mixture containing 2′-OMe A, U, and C, anddeoxy G is referred to as a “dGmH” mixture and aptamers selectedtherefrom are referred to as “dGmH” aptamers. A transcription mixturecontaining deoxy A, and 2′-OMe C, G and U is referred to as a “dAmB”mixture and aptamers selected therefrom are referred to as “dAmB”aptamers, and a transcription mixture containing all 2′-OH nucleotidesis referred to as a “rN” mixture and aptamers selected therefrom arereferred to as “rN” or “rRrY” aptamers. A “mRmY” aptamer is onecontaining all 2′-OMe nucleotides and is usually derived from a r/mGmHoligonucleotide by post-SELEX replacement, when possible, of any 2′-OHGs with 2′-OMe Gs.

A preferred embodiment includes any combination of 2′-OH, 2′-deoxy and2′-OMe nucleotides. A more preferred embodiment includes any combinationof 2′-deoxy and 2′-OMe nucleotides. An even more preferred embodiment iswith any combination of 2′-deoxy and 2′-OMe nucleotides in which thepyrimidines are 2′-OMe (such as dRmY, mRmY or dGmH).

Incorporation of modified nucleotides into the aptamers of the inventionis accomplished before (pre-) the selection process (e.g., a pre-SELEX™process modification). Optionally, aptamers of the invention in whichmodified nucleotides have been incorporated by pre-SELEX™ processmodification can be further modified by post-SELEX™ process modification(i.e., a post-SELEX™ process modification after a pre-SELEX™modification). Pre-SELEX™ process modifications yield modified nucleicacid ligands with specificity for the SELEX™ target and also improved invivo stability. Post-SELEX™ process modifications, i.e., modification(e.g., truncation, deletion, substitution or additional nucleotidemodifications of previously identified ligands having nucleotidesincorporated by pre-SELEX™ process modification) can result in a furtherimprovement of in vivo stability without adversely affecting the bindingcapacity of the nucleic acid ligand having nucleotides incorporated bypre-SELEX™ process modification.

To generate pools of 2′-modified (e.g., 2′-OMe) RNA transcripts inconditions under which a polymerase accepts 2′-modified NTPs thepreferred polymerase is the Y693F/H784A double mutant or the Y693Fsingle mutant. Other polymerases, particularly those that exhibit a hightolerance for bulky 2′-substituents, may also be used in the presentinvention. Such polymerases can be screened for this capability byassaying their ability to incorporate modified nucleotides under thetranscription conditions disclosed herein.

A number of factors have been determined to be important for thetranscription conditions useful in the methods disclosed herein. Forexample, increases in the yields of modified transcript are observedwhen a leader sequence is incorporated into the 5′ end of a fixedsequence at the 5′ end of the DNA transcription template, such that atleast about the first 6 residues of the resultant transcript are allpurines.

Another important factor in obtaining transcripts incorporating modifiednucleotides is the presence or concentration of 2′-OH GTP. Transcriptioncan be divided into two phases: the first phase is initiation, duringwhich an NTP is added to the 3′-hydroxyl end of GTP (or anothersubstituted guanosine) to yield a dinucleotide which is then extended byabout 10-12 nucleotides; the second phase is elongation, during whichtranscription proceeds beyond the addition of the first about 10-12nucleotides. It has been found that small amounts of 2′-OH GTP added toa transcription mixture containing an excess of 2′-OMe GTP aresufficient to enable the polymerase to initiate transcription using2′-OH GTP, but once transcription enters the elongation phase thereduced discrimination between 2′-OMe and 2′-OH GTP, and the excess of2′-OMe GTP over 2′-OH GTP allows the incorporation of principally the2′-OMe GTP.

Another important factor in the incorporation of 2′-OMe substitutednucleotides into transcripts is the use of both divalent magnesium andmanganese in the transcription mixture. Different combinations ofconcentrations of magnesium chloride and manganese chloride have beenfound to affect yields of 2′-OMe transcripts, the optimum concentrationof the magnesium and manganese chloride being dependent on theconcentration in the transcription reaction mixture of NTPs whichcomplex divalent metal ions. To obtain the greatest yields of maximally2′ substituted OMe transcripts (i.e., all A, C, and U and about 90% of Gnucleotides), concentrations of approximately 5 mM magnesium chlorideand 1.5 mM manganese chloride are preferred when each NTP is present ata concentration of 0.5 mM. When the concentration of each NTP is 1.0 mM,concentrations of approximately 6.5 mM magnesium chloride and 2.0 mMmanganese chloride are preferred. When the concentration of each NTP is2.0 mM, concentrations of approximately 9.6 mM magnesium chloride and2.9 mM manganese chloride are preferred. In any case, departures fromthese concentrations of up to two-fold still give significant amounts ofmodified transcripts.

Priming transcription with GMP or guanosine is also important. Thiseffect results from the specificity of the polymerase for the initiatingnucleotide. As a result, the 5′-terminal nucleotide of any transcriptgenerated in this fashion is likely to be 2′-OH G. The preferredconcentration of GMP (or guanosine) is 0.5 mM and even more preferably 1mM. It has also been found that including PEG, preferably PEG-8000, inthe transcription reaction is useful to maximize incorporation ofmodified nucleotides.

For maximum incorporation of 2′-OMe ATP (100%), UTP (100%), CTP (100%)and GTP (˜90%) (“r/mGmH”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%(w/v), Triton X-100 0.01% (w/v), MgCl₂ 5 mM (6.5 mM where theconcentration of each 2′-OMe NTP is 1.0 mM), MnCl₂ 1.5 mM (2.0 mM wherethe concentration of each 2′-OMe NTP is 1.0 mM), 2′-OMe NTP (each) 500μM (more preferably, 1.0 mM), 2′-OH GTP 30 μM, 2′-OH GMP 500 μM, pH 7.5,Y639F/H784A T7 RNA Polymerase 15 units/ml, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong. As used herein, one unit of the Y639F/H784A mutant T7 RNApolymerase (or any other mutant T7 RNA polymerase specified herein) isdefined as the amount of enzyme required to incorporate 1 nmole of2′-OMe NTPs into transcripts under the r/mGmH conditions. As usedherein, one unit of inorganic pyrophosphatase is defined as the amountof enzyme that will liberate 1.0 mole of inorganic orthophosphate perminute at pH 7.2 and 25° C.

For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP (“rGmH”)into transcripts the following conditions are preferred: HEPES buffer200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-1000.01% (w/v), MgCl₂ 5 mM (9.6 mM where the concentration of each 2′-OMeNTP is 2.0 mM), MnCl₂ 1.5 mM (2.9 mM where the concentration of each2′-OMe NTP is 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably, 2.0mM), pH 7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

For maximum incorporation (100%) of 2′-OMe UTP and CTP (“rRmY”) intotranscripts the following conditions are preferred: HEPES buffer 200 mM,DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01%(w/v), MgCl₂ 5 mM (9.6 mM where the concentration of each 2′-OMe NTP is2.0 mM), MnCl₂ 1.5 mM (2.9 mM where the concentration of each 2′-OMe NTPis 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably, 2.0 mM), pH 7.5,Y639F/H784A T7 RNA Polymerase 15 units/ml, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong.

For maximum incorporation (100%) of deoxy ATP and GTP. and 2′-OMe UTPand CTP (“dRmY”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermine 2 mM, spermidine 2mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂2.9 mM, 2′-OMe NTP (each) 2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 15units/ml, inorganic pyrophosphatase 5 units/ml, and an all-purine leadersequence of at least 8 nucleotides long.

For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP and 2′-F GTP(“fGmH”) into transcripts the following conditions are preferred: HEPESbuffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), TritonX-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂ 2.9 mM, 2′-OMe NTP (each) 2.0 mM,pH 7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong.

For maximum incorporation (100%) of deoxy ATP and 2′-OMe UTP, GTP andCTP (“dAmB”) into transcripts the following conditions are preferred:HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v),Triton X-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂ 2.9 mM, 2′-OMe NTP (each)2.0 mM, pH 7.5, Y639F 17 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

For each of the above (a) transcription is preferably performed at atemperature of from about 20° C. to about 50° C., preferably from about30° C. to 45° C., and more preferably at about 37° C. for a period of atleast two hours and (b) 50-300 nM of a double stranded DNA transcriptiontemplate is used (200 nM template is used in round 1 to increasediversity (300 nM template is used in dRmY transcriptions)), and forsubsequent rounds approximately 50 nM, a 1/10 dilution of an optimizedPCR reaction, using conditions described herein, is used). The preferredDNA transcription templates are described below (where ARC254 and ARC256transcribe under all 2′-OMe conditions and ARC255 transcribes under rRmYconditions).

ARC254 (SEQ ID NO: 99):5′-CATCGATGCTAGTCGTAACGATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ ARC255 (SEQ ID NO: 100):5′-CATGCATCGCGACTGACTAGCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ARC256 (SEQ ID NO: 101):5′-CATCGATCGATCGATCGACAGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′

Under rN transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates(ATP), 2′-OH guanosine triphosphates (GTP), 2′-OH cytidine triphosphates(CTP), and 2′-OH uridine triphosphates (UTP). The modifiedoligonucleotides produced using the rN transcription mixtures of thepresent invention comprise substantially all 2′-OH adenosine, 2′-OHguanosine, 2′-OH cytidine, and 2′-OH uridine. In a preferred embodimentof rN transcription, the resulting modified oligonucleotides comprise asequence where at least 80% of all adenosine nucleotides are 2′-OHadenosine, at least 80% of all guanosine nucleotides are 2′-OHguanosine, at least 80% of all cytidine nucleotides are 2′-OH cytidine,and at least 80% of all uridine nucleotides are 2′-OH uridine. In a morepreferred embodiment of rN transcription, the resulting modifiedoligonucleotides of the present invention comprise a sequence where atleast 90% of all adenosine nucleotides are 2′-01-1 adenosine, at least90% of all guanosine nucleotides are 2′-OH guanosine, at least 90% ofall cytidine nucleotides are 2′-OH cytidine, and at least 90% of alluridine nucleotides are 2′-OH uridine. In a most preferred embodiment ofrN transcription, the modified oligonucleotides of the present inventioncomprise a sequence where 100% of all adenosine nucleotides are 2′-OHadenosine, 100% of all guanosine nucleotides are 2′-OH guanosine, 100%of all cytidine nucleotides are 2′-OH cytidine, and 100% of all uridinenucleotides are 2′-OH uridine.

Under rRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates,2′-OH guanosine triphosphates, 2′-OMe cytidine triphosphates, and 2′-OMeuridine triphosphates. The modified oligonucleotides produced using therRmY transcription mixtures of the present invention comprisesubstantially all 2′-OH adenosine, 2′-OH guanosine, 2′-OMe cytidine and2′-OMe uridine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all adenosinenucleotides are 2′-OH adenosine, at least 80% of all guanosinenucleotides are 2′-OH guanosine, at least 80% of all cytidinenucleotides are 2′-OMe cytidine and at least 80% of all uridinenucleotides are 2′-OMe uridine. In a more preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least90% of all adenosine nucleotides are 2′-OH adenosine, at least 90% ofall guanosine nucleotides are 2′-OH guanosine, at least 90% of allcytidine nucleotides are 2′-OMe cytidine and at least 90% of all uridinenucleotides are 2′-OMe uridine In a most preferred embodiment, theresulting modified oligonucleotides comprise a sequence where 100% ofall adenosine nucleotides are 2′-OH adenosine, 100% of all guanosinenucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are2′-OMe cytidine and 100% of all uridine nucleotides are 2′-OMe uridine.

Under dRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy adenosinetriphosphates, 2′-deoxy guanosine triphosphates, 2′-O-methyl cytidinetriphosphates, and 2′-O-methyl uridine triphosphates. The modifiedoligonucleotides produced using the dRmY transcription conditions of thepresent invention comprise substantially all 2′-deoxy adenosine,2′-deoxy guanosine, 2′-O-methyl cytidine, and 2′-O-methyl uridine. In apreferred embodiment, the resulting modified oligonucleotides of thepresent invention comprise a sequence where at least 80% of alladenosine nucleotides are 2′-deoxy adenosine, at least 80% of allguanosine nucleotides are 2′-deoxy guanosine, at least 80% of allcytidine nucleotides are 2′-O-methyl cytidine, and at least 80% of alluridine nucleotides are 2′-O-methyl uridine. In a more preferredembodiment, the resulting modified oligonucleotides of the presentinvention comprise a sequence where at least 90% of all adenosinenucleotides are 2′-deoxy adenosine, at least 90% of all guanosinenucleotides are 2′-deoxy guanosine, at least 90% of all cytidinenucleotides are 2′-O-methyl cytidine, and at least 90% of all uridinenucleotides are 2′-O-methyl uridine. In a most preferred embodiment, theresulting modified oligonucleotides of the present invention comprise asequence where 100% of all adenosine nucleotides are 2′-deoxy adenosine,100% of all guanosine nucleotides are 2′-deoxy guanosine, 100% of allcytidine nucleotides are 2′-O-methyl cytidine, and 100% of all uridinenucleotides are 2′-O-methyl uridine.

Under rGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH guanosine triphosphates,2′-OMe cytidine triphosphates, 2′-OMe uridine triphosphates, and 2′-OMeadenosine triphosphates. The modified oligonucleotides produced usingthe rGmH transcription mixtures of the present invention comprisesubstantially all 2′-OH guanosine, 2′-OMe cytidine, 2′-OMe uridine, and2′-OMe adenosine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all guanosinenucleotides are 2′-OH guanosine, at least 80% of all cytidinenucleotides are 2′-OMe cytidine, at least 80% of all uridine nucleotidesare 2′-OMe uridine, and at least 80% of all adenosine nucleotides are2′-OMe adenosine. In a more preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 90% of all guanosinenucleotides are 2′-OH guanosine, at least 90% of all cytidinenucleotides are 2′-OMe cytidine, at least 90% of all uridine nucleotidesare 2′-OMe uridine, and at least 90% of all adenosine nucleotides are2′-OMe adenosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all guanosinenucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are2′-OMe cytidine, 100% of all uridine nucleotides are 2′-OMe uridine, and100% of all adenosine nucleotides are 2′-OMe adenosine.

Under r/mGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OMe adenosine triphosphate,2′-OMe cytidine triphosphate, 2′-OMe guanosine triphosphate, 2′-OMeuridine triphosphate and 2′-OH guanosine triphosphate. The resultingmodified oligonucleotides produced using the r/mGmH transcriptionmixtures of the present invention comprise substantially all 2′-OMeadenosine, 2′-OMe cytidine, 2′-OMe guanosine, and 2′-OMe uridine,wherein the population of guanosine nucleotides has a maximum of about10% 2′-OH guanosine. In a preferred embodiment, the resulting r/mGmHmodified oligonucleotides of the present invention comprise a sequencewhere at least 80% of all adenosine nucleotides are 2′-OMe adenosine, atleast 80% of all cytidine nucleotides are 2′-OMe cytidine, at least 80%of all guanosine nucleotides are 2′-OMe guanosine, at least 80% of alluridine nucleotides are 2′-OMe uridine, and no more than about 10% ofall guanosine nucleotides are 2′-OH guanosine. In a more preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 90% of all adenosine nucleotides are 2′-OMe adenosine, atleast 90% of all cytidine nucleotides are 2′-OMe cytidine, at least 90%of all guanosine nucleotides are 2′-OMe guanosine, at least 90% of alluridine nucleotides are 2′-OMe uridine, and no more than about 10% ofall guanosine nucleotides are 2′-OH guanosine. In a most preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere 100% of all adenosine nucleotides are 2′-OMe adenosine, 100% ofall cytidine nucleotides are 2′-OMe cytidine, 90% of all guanosinenucleotides are 2′-OMe guanosine, and 100% of all uridine nucleotidesare 2′-OMe uridine, and no more than about 10% of all guanosinenucleotides are 2′-OH guanosine.

Under fGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OMe adenosine triphosphates,2′-OMe uridine triphosphates, 2′-OMe cytidine triphosphates, and 2′-Fguanosine triphosphates. The modified oligonucleotides produced usingthe fGmH transcription conditions of the present invention comprisesubstantially all 2′-OMe adenosine, 2′-OMe uridine, 2′-OMe cytidine, and2′-F guanosine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all adenosinenucleotides are 2′-OMe adenosine, at least 80% of all uridinenucleotides are 2′-OMe uridine, at least 80% of all cytidine nucleotidesare 2′-OMe cytidine, and at least 80% of all guanosine nucleotides are2′-F guanosine. In a more preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 90% of all adenosinenucleotides are 2′-OMe adenosine, at least 90% of all uridinenucleotides are 2′-OMe uridine, at least 90% of all cytidine nucleotidesare 2′-OMe cytidine, and at least 90% of all guanosine nucleotides are2′-F guanosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all adenosinenucleotides are 2′-OMe adenosine, 100% of all uridine nucleotides are2′-OMe uridine, 100% of all cytidine nucleotides are 2′-OMe cytidine,and 100% of all guanosine nucleotides are 2′-F guanosine.

Under dAmB transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy adenosinetriphosphates, 2′-OMe cytidine triphosphates, guanosine triphosphates,and 2′-OMe uridine triphosphates. The modified oligonucleotides producedusing the dAmB transcription mixtures of the present invention comprisesubstantially all 2′-deoxy adenosine, 2′-OMe cytidine, 2′-OMe guanosine,and 2′-OMe uridine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all adenosinenucleotides are 2′-deoxy adenosine, at least 80% of all cytidinenucleotides are 2′-OMe cytidine, at least 80% of all guanosinenucleotides are 2′-OMe guanosine, and at least 80% of all uridinenucleotides are 2′-OMe uridine. In a more preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least90% of all adenosine nucleotides are 2′-deoxy adenosine, at least 90% ofall cytidine nucleotides are 2′-OMe cytidine, at least 90% of allguanosine nucleotides are 2′-OMe guanosine, and at least 90% of alluridine nucleotides are 2′-OMe uridine. In a most preferred embodiment,the resulting modified oligonucleotides of the present inventioncomprise a sequence where 100% of all adenosine nucleotides are 2′-deoxyadenosine, 100% of all cytidine nucleotides are 2′-OMe cytidine, 100% ofall guanosine nucleotides are 2′-OMe guanosine, and 100% of all uridinenucleotides are 2′-OMe uridine.

In each case, the transcription products can then be used as the libraryin the SELEX™ process to identify aptamers and/or to determine aconserved motif of sequences that have binding specificity to a giventarget. The resulting sequences are already stabilized, eliminating thisstep from the process to arrive at a stabilized aptamer sequence andgiving a more highly stabilized aptamer as a result. Another advantageof the 2′-OMe SELEX™ process is that the resulting sequences are likelyto have fewer 2′-OH nucleotides required in the sequence, possibly none.To the extent 2′OH nucleotides remain they can be removed by performingpost-SELEX modifications.

As described below, lower but still useful yields of transcripts fullyincorporating 2′ substituted nucleotides can be obtained underconditions other than the optimized conditions described above. Forexample, variations to the above transcription conditions include:

The HEPES buffer concentration can range from 0 to 1 M. The presentinvention also contemplates the use of other buffering agents having apKa between 5 and 10 including, for example,Tris(hydroxymethyl)aminomethane.

The DTT concentration can range from 0 to 400 mM. The methods of thepresent invention also provide for the use of other reducing agentsincluding, for example, mercaptoethanol.

The spermidine and/or spermine concentration can range from 0 to 20 mM.

The PEG-8000 concentration can range from 0 to 50% (w/v). The methods ofthe present invention also provide for the use of other hydrophilicpolymer including, for example, other molecular weight PEG or otherpolyalkylene glycols.

The Triton X-100 concentration can range from 0 to 0.1% (w/v). Themethods of the present invention also provide for the use of othernon-ionic detergents including, for example, other detergents, includingother Triton-X detergents.

The MgCl₂ concentration can range from 0.5 mM to 50 mM. The MnCl₂concentration can range from 0.15 mM to 15 mM. Both MgCl₂ and MnCl₂ mustbe present within the ranges described and in a preferred embodiment arepresent in about a 10 to about 3 ratio of MgCl₂:MnCl₂, preferably, theratio is about 3-5:1, more preferably, the ratio is about 3-4:1.

The 2′-OMe NTP concentration (each NTP) can range from 5 μM to 5 mM.

The 2′-OH GTP concentration can range from 0 μM to 300 μM.

The 2′-OH GMP concentration can range from 0 to 5 mM.

The pH can range from pH 6 to pH 9. The methods of the present inventioncan be practiced within the pH range of activity of most polymerasesthat incorporate modified nucleotides. In addition, the methods of thepresent invention provide for the optional use of chelating agents inthe transcription reaction condition including, for example, EDTA, EGTA,and DTT.

The selected aptamers having the highest affinity and specific bindingas demonstrated by biological assays as described in the examples beloware suitable therapeutics for treating conditions in which the C5complement protein is involved in pathogenesis.

Aptamers with Binding Affinity to Complement System Protein C5

Although the complement system has an important role in the maintenanceof health, it has the potential to cause or contribute to disease. Thus,it is desirable to develop inhibitors of the complement system fortherapeutic use. It is particularly desirable to develop inhibitors ofcomplement protein C5 because it is a component of both the classicaland alternative pathways of complement activation cascades (Matis andRollins (1995) Nature Medicine 1(8):839-842). Accordingly, inhibition ofC5 can prevent complement-mediated damage caused by either pathway. Somecomplement system proteins, such as C1q and C3, are important in thenormal defense mechanisms against microorganisms and in the clearance ofimmune components and damaged tissue; however, C5 is relativelyunimportant for these functions. Thus, C5 function can be inhibited forshort or long periods of time without compromising the protective roleof the complement system.

A therapeutic C5 inhibitor is also desirable because inhibiting cleavageof C5 prevents the generation of two potentially damaging complementactivities. First, inhibiting the generation of C5a from the cleavage ofC5 eliminates the major complement chemotactic and vasoactive activity.Second, inhibiting the generation of C5b from the cleavage of C5 blocksassembly of the cytolytic C5b-9 membrane attack complex (“MAC”).Inhibition of C5 cleavage blocks both the C5a and the C5b effects onleukocytes and on tissue such as endothelial cells (Ward (1996) Am. J.Pathol. 149:1079).

Both C5a and the MAC have been implicated in acute and chronicinflammation associated with human disease, and their role in diseasestates has been confirmed in animal models. C5a is required forcomplement and neutrophil dependent lung vascular injury (Ward (1997) J.Lab. Clin. Med. 129:400; Mulligan et al., (1998) J. Clin. Invest.98:503), and is associated with neutrophil and platelet activation inshock and in burn injury (Schmid et al., (1997) Shock 8:119). The MACmediates muscle injury in acute autoimmune myasthenia gravis (Bieseckerand Gomez (1989) J. Immunol. 142:2654), organ rejection intransplantation (Baldwin et al., (1995) Transplantation 59:797; Braueret al., (1995) Transplantation 59:288; Takahashi et al., (1997) Immunol.Res. 16:273), and renal injury in autoimmune glomerulonephritis(Biesecker (1981) J. Exp. Med. 39:1779; Nangaku (1997) Kidney Int.52:1570). Both C5a and the MAC are implicated in acute myocardialischemia (Homeister and Lucchesi (1994) Annu. Rev. Pharmacol. Toxicol.34:17), acute (Bednar et al., (1997) J. Neurosurg. 86:139) and chronicCNS injury (Morgan (1997) Exp. Clin. Immunogenet. 14:19), leukocyteactivation during extracorporeal circulation (Sun et al., (1995) NucleicAcids Res. 23:2909; Spycher and Nydegger (1995) Infushionsther.Transfusionsmed. 22:36) and in tissue injury associated with autoimmunediseases including arthritis and lupus (Wang et al., (1996) Immunology93:8563).

Complement activation has also been implicated in diabetic retinopathy,and can compound or initiate retinal vascular damage (Zhang et al.,(2002) Diabetes 51:3499). Low level constitutive complement activationnormally occurs in the non-diabetic eye, evidenced by the presence ofMAC and complement regulatory proteins in the eyes of non-diabetic rats,indicating that complement dysregulation occurs in diabetic patients(Sohn et al., (2000) IOVS 41:3492). In addition, C5b-9 deposition hasbeen detected in retinal vessels from diabetic human donors where absentfrom non-diabetic human donors (Zhang et al.), reduced expression ofCD55 and CD59 is shown in diabetic retinas (Zhang et al.), and glycatedCD59 is present in urine from diabetic patients, but not non-diabeticpatients (Acosta et al., (2002) PNAS 97, 5450-5455). Additionally, thecomplement and vascular system is known to be activated in type Idiabetes. See, e.g. Hansen, T. K. et al., Diabetes, 53: 1570-1576(2004). C5a activates endothelial cells via interaction with the immuneand complement systems. See, e.g., Albrecht, E. A. et al., Am JPathology, 164: 849-859 (2004). The vascular system is activated inocular diseases including diabetic retinopathy. See, e.g. Gert, V. B. etal., Invest Opthalmol Vis Sci, 43: 1104-1108 (2002). The complementsystem is also activated in diabetic retinopathy. See, See, e.g. Gert,V. B. et al., Invest Opthalmol Vis Sci, 43: 1104-1108 (2002) andBadouin, C et al., Am J Opthalmol, 105:383-388 (1988).

In some embodiments, the materials of the present invention comprise aseries of nucleic acid aptamers of about 15 to about 60 nucleotides inlength which bind specifically to complement protein C5 and whichfunctionally modulate, e.g., block, the activity of complement proteinC5 in in vivo and/or cell-based assays.

Aptamers that are capable of specifically binding and modulatingcomplement protein C5 are set forth herein. These aptamers provide alow-toxicity, safe, and effective modality of treating, amelioratingand/or preventing a variety of complement-related diseases or disordersincluding, for example, complement-related heart disorders (e.g.,myocardial injury; C5 mediated complement complications relating tocoronary artery bypass graft (CABG) surgery such as post-operativebleeding, systemic neutrophil and leukocyte activation, increased riskof myocardial infarction, and increased cognitive dysfunction;restenosis; and C5 mediated complement complications relating topercutaneous coronary intervention), ischemia-reperfusion injury (e.g.,myocardial infarction, stroke, frostbite), complement-relatedinflammatory disorders (e.g., asthma, arthritis, sepsis, and rejectionafter organ transplantation), and complement-related autoimmunedisorders (e.g., myasthenia gravis, systemic lupus erythematosus (SLE)).Other indications for which C5 inhibition is desirable include, forexample, lung inflammation (Mulligan et al. (1998) J. Clin. Invest.98:503), extracorporeal complement activation (Rinder et al. (1995) J.Clin. Invest. 96:1564), antibody-mediated complement activation(Biesecker et al. (1989) J. Immunol. 142:2654), glomerulonephritis andother renal diseases, ocular indications such as C5 mediated oculartissue damage, e.g. diabetic retinopathy, and paroxysomal nocturnalhemoglobinuria. These aptamers may also be used in diagnostics.

These aptamers may include modifications as described herein including,e.g., conjugation to lipophilic or high molecular weight compounds(e.g., PEG), incorporation of a capping moiety, incorporation ofmodified nucleotides, and modifications to the phosphate back bone.

In one embodiment of the invention an isolated, non-naturally occurringaptamer that binds to the C5 complement protein is provided. In someembodiments, the isolated, non-naturally occurring aptamer has adissociation constant (“K_(d)”) for C5 complement protein of less than100 μM, less than 1 μM, less than 500 nM, less than 100 nM, less than 50nM, less than 1 nM, less than 500 μM, less than 100 μM, less than 50 μM.In some embodiments of the invention, the dissociation constant isdetermined by dot blot titration as described in Example 1 below.

In another embodiment, the aptamer of the invention modulates a functionof the C5 complement protein. In another embodiment, the aptamer of theinvention inhibits a C5 function while in another embodiment the aptamerstimulates a function of C5. A C5 complement protein variant as usedherein encompasses variants that perform essentially the same functionas a C5 complement protein function. A C5 complement protein variantpreferably comprises substantially the same structure and in someembodiments comprises 80% sequence identity, more preferably 90%sequence identity, and more preferably 95% sequence identity to theamino acid sequence of the C5 complement protein comprising the aminoacid sequence below (SEQ ID NO: 102) (cited in Haviland et al., JImmunol. 1991 Jan. 1; 146(1):362-8):

1 mgllgilcfl iflgktwgqe qtyvisapki frvgaseniv iqvygyteaf datisiksyp 61dkkfsyssgh vhlssenkfq nsailtiqpk qlpggqnpvs yvylevvskh fskskrmpit 121ydngflfiht dkpvytpdqs vkvrvyslnd dlkpakretv ltfidpegse vdmveeidhi 181giisfpdfki psnprygmwt ikakykedfs ttgtayfevk eyvlphfsvs iepeynfigy 241knfknfeiti karyfynkvv teadvyitfg iredlkddqk emmqtamqnt mlingiaqvt 301fdsetavkel syysledlnn kylyiavtvi estggfseea eipgikyvls pyklnlvatp 361lflkpgipyp ikvqvkdsld qlvggvpvtl naqtidvnge tsdldpsksv trvddgvasf 421vlnlpsgvtv lefnvktdap dlpeengare gyraiayssl sqsylyidwt dnhkallvge 481hlniivtpks pyidkithyn ylilskgkii hfgtrekfsd asyqsinipv tqnmvpssrl 541lvyyivtgeq taelvsdsvw lnieekcgnq lqvhlspdad ayspgqtvsl nmatgmdswv 601alaavdsavy gvqrgakkpl ervfqfleks dlgcgagggl nnanvfhlag ltfltnanad 661dsqendepck eilrprrtlq kkieeiaaky khsvvkkccy dgacvnndet ceqraarisl 721gprcikafte ccvvasqlra nishkdmqlg rlhmktllpv skpeirsyfp eswlwevhlv 781prrkqlqfal pdslttweiq gvgisntgic vadtvkakvf kdvflemnip ysvvrgeqiq 841lkgtvynyrt sgmqfcvkms avegictses pvidhqgtks skcvrqkveg ssshlvtftv 901lpleiglhni nfsletwfgk eilvktlrvv pegvkresys gvtldprgiy gtisrrkefp 961yripldlvpk teikrilsvk gllvgeilsa vlsqeginil thlpkgsaea elmsvvpvfy 1021vfhyletgnh wnifhsdpli ekqklkkkik egmlsimsyr nadysysvwk ggsastwlta 1081falrvlgqvn kyveqnqnsi cnsllwlven yqldngsfke nsqyqpiklq gtlpvearen 1141slyltaftvi girkafdicp lvkidtalik adnfllentl paqstftlai sayalslgdk 1201thpqfrsivs alkrealvkg nppiyrfwkd nlqhkdssvp ntgtarmvet tayalltsln 1261lkdinyvnpv ikwlseeqry gggfystqdt inaiegltey sllvkqlrls mdidvsykhk 1321galhnykmtd knflgrpvev llnddlivst gfgsglatvh vttvvhktst seevcsfylk 1381idtqdieash yrgygnsdyk rivacasykp sreesssgss havmdislpt gisaneedlk 1441alvegvdqlf tdyqikdghv ilqlnsipss dflcvrfrif elfevgflsp atftvyeyhr 1501pdkqctmfys tsnikiqkvc egaackcvea dcgqmqeeld ltisaetrkq tackpeiaya 1561ykvsitsitv envfvkykat lldiyktgea vaekdseitf ikkvtctnae lvkgrqylim 1621gkealgikyn fsfryiypld sltwieywpr dttcsscqaf lanldefaed iflngc

In some embodiments of the invention, the sequence identity of targetvariants is determined using BLAST as described below. The terms“sequence identity” in the context of two or more nucleic acid orprotein sequences, refer to two or more sequences or subsequences thatare the same or have a specified percentage of amino acid residues ornucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. For sequence comparison,typically one sequence acts as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al., J Mol.Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(hereinafter “NCBI”). The default parameters used in determiningsequence identity using the software available from NCBI, e.g., BLASTN(for nucleotide sequences) and BLASTP (for amino acid sequences) aredescribed in McGinnis et al., Nucleic Acids Res., 32: W20-W25 (2004).

In another embodiment of the invention, the aptamer has substantiallythe same ability to bind C5 complement protein as that of an aptamercomprising any one of: SEQ ID NOS 1-2, 5-67, 75-81, 83 or 88-98 isprovided. In another embodiment of the invention, the aptamer hassubstantially the same structure and ability to bind C5 complementprotein as that of an aptamer comprising any one of: SEQ ID NOS 1-2,5-67, 75-81, 83 or 88-98. In another embodiment, the aptamers of theinvention have a sequence, including any chemical modifications,according to any one of SEQ ID NOS: 2, 5-67, 75-81, 83 or 88-98. Inanother embodiment, the aptamers of the invention are used as an activeingredient in pharmaceutical compositions. In another embodiment, theaptamers or compositions comprising the aptamers of the invention areused to treat a variety of complement-related diseases or disordersincluding any one selected from the group consisting of:complement-related heart disorders (e.g., myocardial injury; C5 mediatedcomplement complications relating to coronary artery bypass graft (CABG)such as post-operative bleeding, systemic neutrophil and leukocyteactivation, increased risk of myocardial infarction and increasedcognitive dysfunction; restenosis; and C5 mediated complementcomplications relating to percutaneous coronary intervention),ischemia-reperfusion injury (e.g., myocardial infarction, stroke,frostbite), complement-related inflammatory disorders (e.g., asthma,arthritis, sepsis, and rejection after organ transplantation), andcomplement-related autoimmune disorders (e.g., myasthenia gravis,systemic lupus erythematosus (SLE), lung inflammation, extracorporealcomplement activation, antibody-mediated complement activation andocular indications such complement mediated ocular tissue damage such asdiabetic retinopathy.

In one embodiment, the anti-C5 aptamers of the invention include amixture of 2′-fluoro modified nucleotides, 2′-OMe modified nucleotides(“2′-OMe”) and 2′-OH purine residues. A descriptive generic sequence(SEQ ID NO: 1) for a modified anti-C5 aptamer is shown below in Table 1,and the structure is shown in FIG. 3A. The vast majority of purities (Aand G) have been modified to 2′-OMe, excluding only two G residues whichremain 2′-OH (residues shown in outline). The circled residues representa subset of pyrimidines that can be simultaneously modified to 2′-Hwithout substantially altering the anti-C5 activity of the aptamer (seeARC330 in Table 1 below (SEQ ID NO: 2, FIG. 3B)). The underlinedresidues shown in FIG. 3A represent pyrimidine residues that can containeither a 2′-fluoro or a 2′-H modification (but not 2′-OMe), while theboxed residues represent pyrimidine residues that can contain either a2′-fluoro or a 2-OMe modification (but not 2′-H). The residues indicatedwith an arrow (→) must contain a 2′-fluoro modification. Without a2′-fluoro modification at the residues indicated by an arrow (→),resulting hemolytic activity of the resulting aptamer is substantiallydecreased. In a preferred embodiment, an anti-C5 aptamer of theinvention comprises a nucleotide sequence according to SEQ ID NO: 1.

An example of an anti-C5 aptamer according to the invention is ARC186(SEQ ID NO: 4) which is shown in FIG. 3C and described in U.S. Pat. No.6,395,888 which is herein incorporated by reference in its entirety. All21 pyrimidine residues of ARC186 have 2′-fluoro modifications. Themajority of purities (14 residues) have 2′-OMe modifications, except forthree 2′-OH purine residues (shown in outline in FIG. 3C). The anti-C5aptamers of the invention can also include different mixtures of2′-fluoro and 2′-H modifications. For example, another anti-C5 aptamerof the invention is the ARC330 (SEQ ID NO: 2) shown in FIG. 3B. ARC330SEQ ID NO: 2) contains seven 2′-H modifications (circled residues inFIG. 3B), 14 pyrimidine residues with 2′-fluoro modifications, 14 purineresidues with 2′-OMe modifications, and three 2′-OH purine residues(shown in outline in FIG. 3B).

Other combinations of aptamers containing a mixture of 2′-fiuoromodifications, 2′-OMe modifications, 2′-OH purine residues, andconjugation to non-immunogenic, high molecular weight compounds (e.g.,PEG) of varying size, each of which were derived from ARC186 (SEQ ID NO:4), are described in Table 1 below. The invention comprises aptamers asdescribed in Table 1 below. The invention also comprises aptamers asdescribed below but lacking the indicated 3′ cap (e.g., inverteddeoxythymidine cap) and/or aptamers indicated below but comprising a 3′cap (e.g., inverted dT) where a 3′ cap is not indicated.

Unless indicated otherwise, the nucleotide sequences in Table 1 beloware listed in the 5′ to 3′ direction. For each of the individualsequences in Table 1, all 2′-OMe purine or pyrimidine modifications areindicated by an “m” preceding the corresponding nucleotide; all2′-fluoro pyrimidine modifications are indicated by an “f’ preceding thecorresponding nucleotide; all purine or pyrimidine deoxy modificationsare indicated by a “d” preceding the corresponding nucleotide; and anypurine or pyrimidine appearing without an “m”, “f”, or “d” preceding thenucleotide indicates a 2′-OH residue. Further a “3T” indicates aninverted deoxy thymidine, “NH” indicates a hexylamine linker, “NH₂”indicates a hexylamine terminal group, “PEG” indicates a polyethyleneglycol group having the indicated molecular weight, and “biotin”indicates an aptamer having biotin conjugated to the 5′ end.

TABLE 1 SEQ ID NO: 1X₁X₂fCfCrGfCX₃X₄fUX₅X₆X₇X₈X₉X₁₀X₁₁rGX₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃fUfUX₂₄X₂₅X₂₆X₂₇X₂₈fCX₂₉ where: X₁ = fC or mCX₂ = rG orgy X₃ = rG or mG X₄ = rG or mG X₅ = fC or dC X₆ = fU or dTX₇ = fC or dC X₈ = rA or mA X₉ = rG or mG X₁₀ = rG or mG X₁₁ = fC or dCX₁₂ = fC or mC X₁₃ = fU or mU X₁₄ = rG or mG X₁₅ = rA or mAX₁₆ = rG or mG X₁₇ = fU or dT X₁₈ = fC or dC X₁₉ = fU or dTX₂₀ = rG or mG X₂₁ = rA or mA X₂₂ = rG or mG X₂₃ = fU or dTX₂₄ = rA or mA X₂₅ = fC or dC X₂₆ = fC or dC X₂₇ = fU or dTX₂₈ = rG or mG X₂₉ = rG or mG ARC330 (SEQ ID NO: 2)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC185 (SEQ ID NO: 3) GAfCGAfUGfCGGfUfCfUfCAfUGfCGfUfCGAGfUGfUGAGfUfUfUAfCfCfUfUfCGfUfC ARC186 (SEQ ID NO: 4)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC187 (SEQ ID NO: 5) 40 kDa PEG-- NH-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T Where the branched 40 kDa PEG is ,3-bis(mPEG-[20 kDa])-propyl-2-(4′-butamide) ARC188 (SEQ ID NO: 6)AGGAfCGAfUGfCGGfUfCfUfCAfUGfCGfUfCGAGfUGfUGAGfUfU fUAfCfCfUfUfCGfUfCARC189 (SEQ ID NO: 7) AGfCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG ARC250 (SEQ ID NO: 8)GGfCGfCfCGfCGGfUfCfUfCAGGfCGfCfUGAGfUfCfUGAGfUfUfU AfCfCfUGfCGARC296 (SEQ ID NO: 9) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAdCdCfUmGfCmG-3T ARC297 (SEQ ID NO: 10)mCmGmCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAdCdCfUmGmCmG-3T ARC331 (SEQ ID NO: 11)dCmGdCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGdCmGARC332 (SEQ ID NO: 12) dCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmG ARC333 (SEQ ID NO: 13)fCmGdCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC334 (SEQ ID NO: 14) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGdCmG ARC411 (SEQ ID NO: 15)fCmGmCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC412 (SEQ ID NO: 16) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCUmGmCmG ARC413 (SEQ ID NO: 17)mCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC414 (SEQ ID NO: 18) mCmGmCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGmCmG ARC415 (SEQ ID NO: 19)fCmGfCdCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC416 (SEQ ID NO: 20) fCmGfCfCGdCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmG ARC417 (SEQ ID NO: 21)fCmGfCdCGdCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC418 (SEQ ID NO: 22) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGdCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmG ARC419 (SEQ ID NO: 23)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCTmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC420 (SEQ ID NO: 24) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGdCTmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmG ARC421 (SEQ ID NO: 25)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGTfUfUAfCfCfUmGfCmGARC422 (SEQ ID NO: 26) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdcTmGmAmGfUTfUAfCfCfUmGfCmG ARC423 (SEQ ID NO: 27)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUTAfCfCfUmGfCmGARC424 (SEQ ID NO: 28) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGTTTAfCfCfUmGfCmG ARC425 (SEQ ID NO: 29)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCTmGfCmGARC426 (SEQ ID NO: 30) fCmGfCfCGfCmGmGmUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAdCdCfUmGfCmG ARC427 (SEQ ID NO: 31)fCmGfCmCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC428 (SEQ ID NO: 32) fCmGfCfCGmCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmG ARC429 (SEQ ID NO: 33)fCmGfCmCGmCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC430 (SEQ ID NO: 34)fCmGfCfCGfCmGmGfUdCfUdCmAmGmGdCGmCfUmGmAmGfUdCfUmGmAmGfUfUfUAfCfCfUmGfCmG ARC431 (SEQ ID NO: 35)fCmGfCfCGfCmGmGfUdCfUdCmAmGmGdCGfCmUmGmAmGfUdCfUmGmAmGfUfUfUAfCfCfUmGfCm ARC432 (SEQ ID NO: 36)fCmGfCfCGfCmGmGfUdCfUdCmAmGmGdCGmCmUmGmAmGfUdCfUmGmAmGfUfUfUAfCfCfUmGfCmG ARC433 (SEQ ID NO: 37)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGmUfUfUAfCfCfUmGfCmGARC434 (SEQ ID NO: 38) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUmUfUAfCfCfUmGfCmG ARC435 (SEQ ID NO: 39)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUmUAfCfCfUmGfCmGARC436 (SEQ ID NO: 40) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGmUmUmUAfCfCfUmGfCmG ARC437 (SEQ ID NO: 41)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCmUmGfCmGARC438 (SEQ ID NO: 42)fCmGfCfCdGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC439 (SEQ ID NO: 43)fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCdGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmGfCmGARC440 (SEQ ID NO: 44) fCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUdAfCfCfUmGfCmG ARC457 (SEQ ID NO: 45)mGfCmGfUfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmAfCmGmC ARC458 (SEQ ID NO: 46)mGmGmGfCgFCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmA mGfUfUfUAfCfCfUmCmCmCARC459 (SEQ ID NO: 47) mGfCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmGmC ARC473 (SEQ ID NO: 48)mGmGmAfCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmGfUfCfU-3T ARC522 (SEQ ID NO: 49)mGmGfCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGmCmUmGmAmGTdCTmGmAmGTfUfUAdCdCTmGfCmGmCmC ARC523 (SEQ ID NO: 50)mGmGmCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGmCmUmGmAmGTdCTmGmAmGTTTAdCdCTmGdCmGmCmC ARC524 (SEQ ID NO: 51)mGmGmCmGdCdCGdCmGmGTdCTdCmAmGmGdCGmCmUmGmAmGTdCTmGmAmGTTTmAdCdCTmGdCmGmCmC ARC525 (SEQ ID NO: 52)mGmGmCmGdCdCGdCmGmGTdCmUmCmAmGmGdCGmCmUmGmAmGmUmCmUmGmAmGTTTmAdCdCTmGdCmGmCmC ARC532 (SEQ ID NO: 53) Biotin-AGfCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG ARC543 (SEQ ID NO: 54)mGmGfCmGfCfCGfCmGmGfUdCTdCmAmGmGdCGfCfUmGmAmGTdCTmGmAmGfUfUfUAfCfCfUmGfCmGmCmC ARC544 (SEQ ID NO: 55)mGmGfCmGfCfCGfCmGmGfUmCmUmCmAmGmGmCGfCfUmGmAmGmUmCmUmGmAmGfUfUfUAfCfCfUmGfCmGmCmC ARC550 (SEQ ID NO: 56)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUmAfCfCfUmGfCmG-3T ARC551 (SEQ ID NO: 57)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGmCmUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC552 (SEQ ID NO: 58)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGTfUfUAfCfCfUmGfCmG-3T ARC553 (SEQ ID NO: 59)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGmCmUmGmAmGfUfCfUmGmAmGfUfUfUmAfCfCfUmGfCmG-3T ARC554 (SEQ ID NO: 60)fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGmCmUmGmAmGfUfCfUmGmAmGTfUfUmAfCfCfUmGfCmG-3T ARC 657 (SEQ ID NO: 61) 20 kDa PEG-NH-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC 658 (SEQ ID NO: 62) 30 kDa PEG-NH-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC 672 (SEQ ID NO: 63) NH2-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC 706 (SEQ ID NO: 64) 10 kDa PEG-NH-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC1537 (SEQ ID NO: 65) 40 kDa PEG-NH-fCmGfCfCGfCmGmGfUfCfUCmAmGmGfCGfCfUmGmAmGfuFcfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T ARC1730) (SEQ ID NO: 66) PEG20K-NH-fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-NH-PEG20K ARC1905 (SEQ ID NO: 67) 40K PEG-NH--fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3TWhere the branched 40 kDa PEG is 2,3-bis(mPEG-[20 kDa])-propyl-1-carbamoyl ARC243 (SEQ ID NO: 68)GGfCGAfUfUAfCfUGGGAfCGGAfCfUfCGfCGAfUGfUGAGfCfCfCA GAfCGAfCfUfCGfCfCARC244 (SEQ ID NO: 69) GGfCfUfUfCfUGAAGAfUfUAfUfUfUfCGfCGAfUGfUGAAfCfUfCfCAGAfCfCfCfC

Other aptamers of the invention that bind complement protein C5 aredescribed below in Example 3.

In some embodiments aptamer therapeutics of the present invention havegreat affinity and specificity to their targets while reducing thedeleterious side effects from non-naturally occurring nucleotidesubstitutions if the aptamer therapeutics break down in the body ofpatients or subjects. In some embodiments, the therapeutic compositionscontaining the aptamer therapeutics of the present invention are free ofor have a reduced amount of fluorinated nucleotides.

The aptamers of the present invention can be synthesized using anyoligonucleotide synthesis techniques known in the art including solidphase oligonucleotide synthesis techniques well known in the art (see,e.g., Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986) and Froehleret al., Tet. Lett. 27:5575-5578 (1986)) and solution phase methods suchas triester synthesis methods (see, e.g., Sood et al., Nucl. Acid Res.4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449 (1978)).

Pharmaceutical Compositions

The invention also includes pharmaceutical compositions containingaptamer molecules that bind to complement protein C5. In someembodiments, the compositions are suitable for internal use and includean effective amount of a pharmacologically active compound of theinvention, alone or in combination, with one or more pharmaceuticallyacceptable carriers. The compounds are especially useful in that theyhave very low, if any toxicity.

Compositions of the invention can be used to treat or prevent apathology, such as a disease or disorder, or alleviate the symptoms ofsuch disease or disorder in a patient. For example, compositions of thepresent invention can be used to treat or prevent a pathology associatedwith complement-related heart disorders (e.g., myocardial injury; C5mediated complement complications relating to coronary artery bypassgraft (CABG) surgery such as post-operative bleeding, systemicneutrophil and leukocyte activation, increased risk of myocardialinfarction and increased cognitive dysfunction; restenosis; and C5mediated complications relating to percutaneous coronary intervention);ischemia-reperfusion injury (e.g., myocardial infarction, stroke,frostbite); complement-related inflammatory disorders (e.g., asthma,arthritis, sepsis, and rejection after organ transplantation); andcomplement-related autoimmune disorders (e.g., myasthenia gravis,systemic lupus erythematosus (SLE, or lupus); lung inflammation;extracorporeal complement activation; antibody-mediated complementactivation; and ocular indications such as diabetic retinopathy.Compositions of the invention are useful for administration to a subjectsuffering from, or predisposed to, a disease or disorder which isrelated to or derived from complement protein C5 to which the aptamersof the invention specifically bind.

Compositions of the invention can be used in a method for treating apatient or subject having a pathology. The methods of the inventioninvolve administering to the patient or subject an aptamer or acomposition comprising aptamers that bind to complement protein C5, sothat binding of the aptamer to complement protein C5 alters itsbiological function, thereby treating the pathology.

The patient or subject having a pathology, i.e., the patient or subjecttreated by the methods of this invention can be a vertebrate, moreparticularly a mammal, or more particularly, a human.

In practice, the aptamers or their pharmaceutically acceptable salts,are administered in amounts which will be sufficient to exert theirdesired biological activity, e.g., inhibiting the binding of the aptamertarget to its receptor, preventing cleavage of a target protein.

One aspect of the invention comprises an aptamer composition of theinvention in combination with other treatments for C5 mediatedcomplement disorders. The aptamer composition of the invention maycontain, for example, more than one aptamer. In some examples, anaptamer composition of the invention, containing one or more compoundsof the invention, is administered in combination with another usefulcomposition such as an anti-inflammatory agent, an immunosuppressant, anantiviral agent, or the like. Furthermore, the compounds of theinvention may be administered in combination with a cytotoxic,cytostatic, or chemotherapeutic agent such as an alkylating agent,anti-metabolite, mitotic inhibitor or cytotoxic antibiotic, as describedabove. In general, the currently available dosage forms of the knowntherapeutic agents for use in such combinations will be suitable.

“Combination therapy” (or “co-therapy”) includes the administration ofan aptamer composition of the invention and at least a second agent aspart of a specific treatment regimen intended to provide the beneficialeffect from the co-action of these therapeutic agents. The beneficialeffect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected).

“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner, Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administeredtopically or all therapeutic agents may be administered by injection.The sequence in which the therapeutic agents are administered is notnarrowly critical unless noted otherwise. “Combination therapy” also canembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients. Wherethe combination therapy further comprises a non-drug treatment, thenon-drug treatment may be conducted at any suitable time so long as abeneficial effect from the co-action of the combination of thetherapeutic agents and non-drug treatment is achieved. For example, inappropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

Therapeutic or pharmacological compositions of the present inventionwill generally comprise an effective amount of the active component(s)of the therapy, dissolved or dispersed in a pharmaceutically acceptablemedium. Pharmaceutically acceptable media or carriers include any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Supplementary active ingredients can also be incorporatedinto the therapeutic compositions of the present invention.

The preparation of pharmaceutical or pharmacological compositions willbe known to those of skill in the art in light of the presentdisclosure. Typically, such compositions may be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection; as tablets orother solids for oral administration; as time release capsules; or inany other form currently used, including eye drops, creams, lotions,salves, inhalants and the like. The use of sterile formulations, such assaline-based washes, by surgeons, physicians or health care workers totreat a particular area in the operating field may also be particularlyuseful. Compositions may also be delivered via microdevice,microparticle or sponge.

Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the host animal to be treated.Precise amounts of active compound required for administration depend onthe judgment of the practitioner and are peculiar to each individual.

A minimal volume of a composition required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

For instance, for oral administration in the form of a tablet or capsule(e.g., a gelatin capsule), the active drug component can be combinedwith an oral, non-toxic pharmaceutically acceptable inert carrier suchas ethanol, glycerol, water and the like. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, magnesium aluminum silicate, starch paste,gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, natural sugars such as glucose or beta-lactose,corn sweeteners, natural and synthetic gums such as acacia, tragacanthor sodium alginate, polyethylene glycol, waxes and the like. Lubricantsused in these dosage forms include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gumstarches, agar, alginic acid or its sodium salt, or effervescentmixtures, and the like. Diluents, include, e.g., lactose, dextrose,sucrose, mannitol, sorbitol, cellulose and/or glycine.

Injectable compositions are preferably aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and typically contain about 0.1 to 75%, preferably about 1to 50%, of the active ingredient.

The compounds of the invention can also be administered in such oraldosage forms as timed release and sustained release tablets or capsules,pills, powders, granules, elixirs, tinctures, suspensions, syrups andemulsions.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.Additionally, solid forms suitable for dissolving in liquid prior toinjection can be formulated.

The compounds of the present invention can be administered inintravenous (both bolus and infusion), intraperitoneal, subcutaneous orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, according to U.S. Pat. No.3,710,795, incorporated herein by reference.

Furthermore, preferred compounds for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, inhalants, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Other preferred topicalpreparations include creams, ointments, lotions, aerosol sprays andgels, wherein the concentration of active ingredient would typicallyrange from 0.01% to 15%, w/w or w/v.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like may beused. The active compound defined above, may be also formulated assuppositories using for example, polyalkylene glycols, for example,propylene glycol, as the carrier. In some embodiments, suppositories areadvantageously prepared from fatty emulsions or suspensions.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, containing cholesterol,stearylamine or phosphatidylcholines. In some embodiments, a film oflipid components is hydrated with an aqueous solution of drug to a formlipid layer encapsulating the drug, as described in U.S. Pat. No.5,262,564. For example, the aptamer molecules described herein can beprovided as a complex with a lipophilic compound or non-immunogenic,high molecular weight compound constructed using methods known in theart. An example of nucleic-acid associated complexes is provided in U.S.Pat. No. 6,011,020.

The compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances such asfor example, sodium acetate, and triethanolamine oleate.

The dosage regimen utilizing the aptamers is selected in accordance witha variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular aptamer or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.05 to 7500 mg/day orally. Thecompositions are preferably provided in the form of scored tabletscontaining 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0,500.0 and 1000.0 mg of active ingredient. Compounds of the presentinvention may be administered in a single daily dose, or the total dailydosage may be administered in divided doses of two, three or four timesdaily.

Infused dosages, intranasal dosages and transdermal dosages will rangebetween 0.05 to 7500 mg/day. Subcutaneous, intravenous and intraperinealdosages will range between 0.05 to 3800 mg/day.

Effective plasma levels of the compounds of the present invention rangefrom 0.002 mg/mL to 50 mg/mL.

Modulation of Pharmacokinetics and Biodistribution of AptamerTherapeutics

It is important that the pharmacokinetic properties for alloligonucleotide-based therapeutics, including aptamers, be tailored tomatch the desired pharmaceutical application. While aptamers directedagainst extracellular targets do not suffer from difficulties associatedwith intracellular delivery (as is the case with antisense andRNAi-based therapeutics), such aptamers must still be able to bedistributed to target organs and tissues, and remain in the body(unmodified) for a period of time consistent with the desired dosingregimen.

Thus, the present invention provides materials and methods to affect thepharmacokinetics of aptamer compositions, and, in particular, theability to tune aptamer pharmacokinetics. The tunability of (i.e., theability to modulate) aptamer pharmacokinetics is achieved throughconjugation of modifying moieties (e.g., PEG polymers) to the aptamerand/or the incorporation of modified nucleotides (e.g., 2′-fluoro or2′-OMe) to alter the chemical composition of the nucleic acid. Theability to tune aptamer pharmacokinetics is used in the improvement ofexisting therapeutic applications, or alternatively, in the developmentof new therapeutic applications. For example, in some therapeuticapplications, e.g., in anti-neoplastic or acute care settings whererapid drug clearance or turn-off may be desired, it is desirable todecrease the residence times of aptamers in the circulation.Alternatively, in other therapeutic applications, e.g., maintenancetherapies where systemic circulation of a therapeutic is desired, it maybe desirable to increase the residence times of aptamers in circulation.

In addition, the tunability of aptamer pharmacokinetics is used tomodify the biodistribution of an aptamer therapeutic in a subject. Forexample, in some therapeutic applications, it may be desirable to alterthe biodistribution of an aptamer therapeutic in an effort to target aparticular type of tissue or a specific organ (or set of organs). Inthese applications, the aptamer therapeutic preferentially accumulatesin a specific tissue or organ(s). In other therapeutic applications, itmay be desirable to target tissues displaying a cellular marker or asymptom associated with a given disease, cellular injury or otherabnormal pathology, such that the aptamer therapeutic preferentiallyaccumulates in the affected tissue. For example, as described incopending provisional application U.S. Ser. No. 60/550,790, filed onMar. 5, 2004, and entitled “Controlled Modulation of thePharmacokinetics and Biodistribution of Aptamer Therapeutics),PEGylation of an aptamer therapeutic (e.g., PEGylation with a 20 kDa PEGpolymer) is used to target inflamed tissues, such that the PEGylatedaptamer therapeutic preferentially accumulates in inflamed tissue.

To determine the pharmacokinetic and biodistribution profiles of aptamertherapeutics (e.g., aptamer conjugates or aptamers having alteredchemistries, such as modified nucleotides) a variety of parameters aremonitored. Such parameters include, for example, the half-life(t_(1/2)), the plasma clearance (Cl), the volume of distribution (Vss),the area under the concentration-time curve (AUC), maximum observedserum or plasma concentration (C_(max)), and the mean residence time(MRT) of an aptamer composition. As used herein, the term “AUC” refersto the area under the plot of the plasma concentration of an aptamertherapeutic versus the time after aptamer administration. The AUC valueis used to estimate the bioavailability (i.e., the percentage ofadministered aptamer therapeutic in the circulation after aptameradministration) and/or total clearance (Cl) (i.e., the rate at which theaptamer therapeutic is removed from circulation) of a given aptamertherapeutic. The volume of distribution relates the plasma concentrationof an aptamer therapeutic to the amount of aptamer present in the body.The larger the Vss, the more an aptamer is found outside of the plasma(i.e., the more extravasation).

The present invention provides materials and methods to modulate, in acontrolled manner, the pharmacokinetics and biodistribution ofstabilized aptamer compositions in vivo by conjugating an aptamer to amodulating moiety such as a small molecule, peptide, or polymer terminalgroup, or by incorporating modified nucleotides into an aptamer. Asdescribed herein, conjugation of a modifying moiety and/or alteringnucleotide(s) chemical composition alters fundamental aspects of aptamerresidence time in circulation and distribution to tissues.

In addition to clearance by nucleases, oligonucleotide therapeutics aresubject to elimination via renal filtration. As such, anuclease-resistant oligonucleotide administered intravenously typicallyexhibits an in vivo half-life of <10 min, unless filtration can beblocked. This can be accomplished by either facilitating rapiddistribution out of the blood stream into tissues or by increasing theapparent molecular weight of the oligonucleotide above the effectivesize cut-off for the glomerulus. Conjugation of small therapeutics to aPEG polymer (PEGylation), described below, can dramatically lengthenresidence times of aptamers in circulation, thereby decreasing dosingfrequency and enhancing effectiveness against vascular targets.

Aptamers can be conjugated to a variety of modifying moieties, such ashigh molecular weight polymers, e.g., PEG; peptides, e.g., Tat (a13-amino acid fragment of the HIV Tat protein (Vives, et al. (1997), J.Biol. Chem. 272(25): 16010-7)), Ant (a 16-amino acid sequence derivedfrom the third helix of the Drosophila antennapedia homeotic protein(Pietersz, et al. (2001), Vaccine 19(11-12): 1397-405)) and Arg7 (ashort, positively charged cell-permeating peptides composed ofpolyarginine (Arg7) (Rothbard, et al. (2000), Nat. Med. 6(11): 1253-7;Rothbard, J et al. (2002), J. Med. Chem. 45(17): 3612-8)); and smallmolecules, e.g., lipophilic compounds such as cholesterol. Among thevarious conjugates described herein, in vivo properties of aptamers arealtered most profoundly by complexation with PEG groups. For example,complexation of a mixed 2′F and 2′-OMe modified aptamer therapeutic witha 20 kDa PEG polymer hinders renal filtration and promotes aptamerdistribution to both healthy and inflamed tissues. Furthermore, the 20kDa PEG polymer-aptamer conjugate proves nearly as effective as a 40 kDaPEG polymer in preventing renal filtration of aptamers. While one effectof PEGylation is on aptamer clearance, the prolonged systemic exposureafforded by presence of the 20 kDa moiety also facilitates distributionof aptamer to tissues, particularly those of highly perfused organs andthose at the site of inflammation. The aptamer-20 kDa PEG polymerconjugate directs aptamer distribution to the site of inflammation, suchthat the PEGylated aptamer preferentially accumulates in inflamedtissue. In some instances, the 20 kDa PEGylated aptamer conjugate isable to access the interior of cells, such as, for example, kidneycells.

Overall, effects on aptamer pharmacokinetics and tissue distributionproduced by low molecular weight modifying moieties, includingcholesterol and cell-permeating peptides are less pronounced than thoseproduced as a result of PEGylation or modification of nucleotides (e.g.,an altered chemical composition). While not intending to be bound bytheory, it is suggested that cholesterol-mediated associations withplasma lipoproteins, postulated to occur in the case of the antisenseconjugate, are precluded in the particular context of theaptamer-cholesterol conjugate folded structure, and/or relate to aspectof the lipophilic nature of the cholesterol group. Like cholesterol, thepresence of a Tat peptide tag promotes clearance of aptamer from theblood stream, with comparatively high levels of conjugate appearing inthe kidneys at 48 hrs. Other peptides (e.g., Ant, Arg₇) that have beenreported in the art to mediate passage of macromolecules across cellularmembranes in vitro, do not appear to promote aptamer clearance fromcirculation. However, like Tat, the Ant conjugate significantlyaccumulates in the kidneys relative to other aptamers. While notintending to be bound by theory, it is possible that unfavorablepresentation of the Ant and Arg₇ peptide modifying moieties in thecontext of three dimensionally folded aptamers in vivo impairs theability of these peptides to influence aptamer transport properties.

Modified nucleotides can also be used to modulate the plasma clearanceof aptamers. For example, an unconjugated aptamer which incorporatesboth 2′-F and 2′-OMe stabilizing chemistries, which is typical ofcurrent generation aptamers as it exhibits a high degree of nucleasestability in vitro and in vivo, displays rapid loss from plasma (i.e.,rapid plasma clearance) and a rapid distribution into tissues, primarilyinto the kidney, when compared to unmodified aptamer.

PEG-Derivatized Nucleic Acids

As described above, derivatization of nucleic acids with high molecularweight non-immunogenic polymers has the potential to alter thepharmacokinetic and pharmacodynamic properties of nucleic acids makingthem more effective therapeutic agents. Favorable changes in activitycan include increased resistance to degradation by nucleases, decreasedfiltration through the kidneys, decreased exposure to the immune system,and altered distribution of the therapeutic through the body.

The aptamer compositions of the invention may be derivatized withpolyalkylene glycol (“PAG”) moieties. Examples of PAG-derivatizednucleic acids are found in U.S. patent application Ser. No. 10/718,833,filed on Nov. 21, 2003, which is herein incorporated by reference in itsentirety. Typical polymers used in the invention include poly(ethyleneglycol) (“PEG’), also known as poly(ethylene oxide) (“PEO”) andpolypropylene glycol (including poly isopropylene glycol). Additionally,random or block copolymers of different alkylene oxides (e.g., ethyleneoxide and propylene oxide) can be used in many applications. In its mostcommon form, a polyalkylene glycol, such as PEG, is a linear polymerterminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH. This polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can also be represented asHO-PEG-OH, where it is understood that the -PEG- symbol represents thefollowing structural unit: —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂— where ntypically ranges from about 4 to about 10,000.

As shown, the PEG molecule is di-functional and is sometimes referred toas “PEG diol.” The terminal portions of the PEG molecule are relativelynon-reactive hydroxyl moieties, the —OH groups, that can be activated,or converted to functional moieties, for attachment of the PEG to othercompounds at reactive sites on the compound. Such activated PEG diolsare referred to herein as bi-activated PEGs. For example, the terminalmoieties of PEG diol have been functionalized as active carbonate esterfor selective reaction with amino moieties by substitution of therelatively nonreactive hydroxyl moieties, —OH, with succinimidyl activeester moieties from N-hydroxy succinimide.

In many applications, it is desirable to cap the PEG molecule on one endwith an essentially non-reactive moiety so that the PEG molecule ismono-functional (or mono-activated). In the case of protein therapeuticswhich generally display multiple reaction sites for activated PEGs,bi-functional activated PEGs lead to extensive cross-linking, yieldingpoorly functional aggregates. To generate mono-activated PEGs, onehydroxyl moiety on the terminus of the PEG diol molecule typically issubstituted with non-reactive methoxy end moiety, —OCH₃. The other,un-capped terminus of the PEG molecule typically is converted to areactive end moiety that can be activated for attachment at a reactivesite on a surface or a molecule such as a protein.

PAGs are polymers which typically have the properties of solubility inwater and in many organic solvents, lack of toxicity, and lack ofimmunogenicity. One use of PAGs is to covalently attach the polymer toinsoluble molecules to make the resulting PAG-molecule “conjugate”soluble. For example, it has been shown that the water-insoluble drugpaclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, etal., J. Org. Chem., 60:331-336 (1995). PAG conjugates are often used notonly to enhance solubility and stability but also to prolong the bloodcirculation half-life of molecules.

Polyalkylated compounds of the invention are typically between 5 and 80kD in size however any size can be used, the choice dependent on theaptamer and application. Other PAG compounds of the invention arebetween 10 and 80 kD in size. Still other PAG compounds of the inventionare between 10 and 60 kD in size. For example, a PAG polymer may be atleast 10, 20, 30, 40, 50, 60, or 80 kD in size. Such polymers can belinear or branched.

In contrast to biologically-expressed protein therapeutics, nucleic acidtherapeutics are typically chemically synthesized from activated monomernucleotides. PEG-nucleic acid conjugates may be prepared byincorporating the PEG using the same iterative monomer synthesis. Forexample, PEGs activated by conversion to a phosphoramidite form can beincorporated into solid-phase oligonucleotide synthesis. Alternatively,oligonucleotide synthesis can be completed with site-specificincorporation of a reactive PEG attachment site. Most commonly this hasbeen accomplished by addition of a free primary amine at the 5′-terminus(incorporated using a modified phosphoramidite in the last coupling stepof solid phase synthesis). Using this approach, a reactive PEG (e.g.,one which is activated so that it will react and form a bond with anamine) is combined with the purified oligonucleotide and the couplingreaction is carried out in solution. In some embodiment the polymers arebranched PEG molecules. In still other embodiments the polymers are 40kDa branched PEG, see, e.g. (1,3-bis(mPEG-[20kDa])-propyl-2-(4′-butamide) depicted in FIG. 4. In some embodiments the40 kD branched PEG (1,3-bis(mPEG-[20 kDa])-propyl-2-(4′-butamide) isattached to the 5′ end of the aptamer as depicted in FIG. 5.

The ability of PEG conjugation to alter the biodistribution of atherapeutic is related to a number of factors including the apparentsize (e.g., as measured in terms of hydrodynamic radius) of theconjugate. Larger conjugates (>10 kDa) are known to more effectivelyblock filtration via the kidney and to consequently increase the serumhalf-life of small macromolecules (e.g., peptides, antisenseoligonucleotides). The ability of PEG conjugates to block filtration hasbeen shown to increase with PEG size up to approximately 50 kDa (furtherincreases have minimal beneficial effect as half life becomes defined bymacrophage-mediated metabolism rather than elimination via the kidneys).

Production of high molecular weight PEGs (>10 kDa) can be difficult,inefficient, and expensive. As a route towards the synthesis of highmolecular weight PEG-nucleic acid conjugates, previous work has beenfocused towards the generation of higher molecular weight activatedPEGs. One method for generating such molecules involves the formation ofa branched activated PEG in which two or more PEGs are attached to acentral core carrying the activated group. The terminal portions ofthese higher molecular weight PEG molecules, i.e., the relativelynon-reactive hydroxyl (—OH) moieties, can be activated, or converted tofunctional moieties, for attachment of one or more of the PEGs to othercompounds at reactive sites on the compound. Branched activated PEGswill have more than two termini, and in cases where two or more terminihave been activated, such activated higher molecular weight PEGmolecules are referred to herein as, multi-activated PEGs. In somecases, not all termini in a branch PEG molecule are activated. In caseswhere any two termini of a branch PEG molecule are activated, such PEGmolecules are referred to as bi-activated PEGs. In some cases where onlyone terminus in a branch PEG molecule is activated, such PEG moleculesare referred to as mono-activated. As an example of this approach,activated PEG prepared by the attachment of two monomethoxy PEGs to alysine core which is subsequently activated for reaction has beendescribed (Harris et al., Nature, vol. 2: 214-221, 2003).

The present invention provides another cost effective route to thesynthesis of high molecular weight PEG-nucleic acid (preferably,aptamer) conjugates including multiply PEGylated nucleic acids. Thepresent invention also encompasses PEG-linked multimericoligonucleotides, e.g., dimerized aptamers. The present invention alsorelates to high molecular weight compositions where a PEG stabilizingmoiety is a linker which separates different portions of an aptamer,e.g., the PEG is conjugated within a single aptamer sequence, such thatthe linear arrangement of the high molecular weight aptamer compositionis, e.g., nucleic acid-PEG-nucleic acid (-PEG nucleic acid)_(n), where nis greater than or equal to 1.

High molecular weight compositions of the invention include those havinga molecular weight of at least 10 kD. Compositions typically have amolecular weight between 10 and 80 kD in size. High molecular weightcompositions of the invention are at least 10, 20, 30, 40, 50, 60, or 80kD in size.

A stabilizing moiety is a molecule, or portion of a molecule, whichimproves pharmacokinetic and pharmacodynamic properties of the highmolecular weight aptamer compositions of the invention. In some cases, astabilizing moiety is a molecule or portion of a molecule which bringstwo or more aptamers, or aptamer domains, into proximity, or providesdecreased overall rotational freedom of the high molecular weightaptamer compositions of the invention. A stabilizing moiety can be apolyalkylene glycol, such a polyethylene glycol, which can be linear orbranched, a homopolymer or a heteropolymer. Other stabilizing moietiesinclude polymers such as peptide nucleic acids (PNA). Oligonucleotidescan also be stabilizing moieties; such oligonucleotides can includemodified nucleotides, and/or modified linkages, such asphosphorothioates. A stabilizing moiety can be an integral part of anaptamer composition, i.e., it is covalently bonded to the aptamer.

Compositions of the invention include high molecular weight aptamercompositions in which two or more nucleic acid moieties are covalentlyconjugated to at least one polyalkylene glycol moiety. The polyalkyleneglycol moieties serve as stabilizing moieties. In compositions where apolyalkylene glycol moiety is covalently bound at either end to anaptamer, such that the polyalkylene glycol joins the nucleic acidmoieties together in one molecule, the polyalkylene glycol is said to bea linking moiety. In such compositions, the primary structure of thecovalent molecule includes the linear arrangement nucleicacid-PAG-nucleic acid. One example is a composition having the primarystructure nucleic acid-PEG-nucleic acid. Another example is a lineararrangement of nucleic acid PEG—nucleic acid—PEG nucleic acid.

To produce the nucleic acid-PEG-nucleic acid conjugate, the nucleic acidis originally synthesized such that it bears a single reactive site(e.g., it is mono-activated). In a preferred embodiment, this reactivesite is an amino group introduced at the 5′-terminus by addition of amodified phosphoramidite as the last step in solid phase synthesis ofthe oligonucleotide. Following deprotection and purification of themodified oligonucleotide, it is reconstituted at high concentration in asolution that minimizes spontaneous hydrolysis of the activated PEG. Ina preferred embodiment, the concentration of oligonucleotide is 1 mM andthe reconstituted solution contains 200 mM NaHCO₃-buffer, pH 8.3.Synthesis of the conjugate is initiated by slow, step-wise addition ofhighly purified bi-functional PEG. In a preferred embodiment, the PEGdiol is activated at both ends (bi-activated) by derivatization withsuccinimidyl propionate. Following reaction, the PEG-nucleic acidconjugate is purified by gel electrophoresis or liquid chromatography toseparate fully-, partially-, and un-conjugated species. Multiple PAGmolecules concatenated (e.g., as random or block copolymers) or smallerPAG chains can be linked to achieve various lengths (or molecularweights). Non-PAG linkers can be used between PAG chains of varyinglengths.

The 2′-OMe, 2′-fluoro and other modified nucleotide modificationsstabilize the aptamer against nucleases and increase its half life invivo. The 3′-3′-dT cap also increases exonuclease resistance. See, e.g.,U.S. Pat. Nos. 5,674,685; 5,668,264; 6,207,816; and 6,229,002, each ofwhich is incorporated by reference herein in its entirety.

PAG-Derivatization of a Reactive Nucleic Acid

High molecular weight PAG-nucleic acid-PAG conjugates can be prepared byreaction of a mono-functional activated PEG with a nucleic acidcontaining more than one reactive site. In one embodiment, the nucleicacid is bi-reactive, or bi-activated, and contains two reactive sites: a5′-amino group and a 3′-amino group introduced into the oligonucleotidethrough conventional phosphoramidite synthesis, for example:3′-5′-di-PEGylation as illustrated in FIG. 6. In alternativeembodiments, reactive sites can be introduced at internal positions,using for example, the 5-position of pyrimidines, the 8-position ofpurines, or the 2′-position of ribose as sites for attachment of primaryamines. In such embodiments, the nucleic acid can have several activatedor reactive sites and is said to be multiply activated. Followingsynthesis and purification, the modified oligonucleotide is combinedwith the mono-activated PEG under conditions that promote selectivereaction with the oligonucleotide reactive sites while minimizingspontaneous hydrolysis. In the preferred embodiment, monomethoxy-PEG isactivated with succinimidyl propionate and the coupled reaction iscarried out at pH 8.3. To drive synthesis of the bi-substituted PEG,stoichiometric excess PEG is provided relative to the oligonucleotide.Following reaction, the PEG-nucleic acid conjugate is purified by gelelectrophoresis or liquid chromatography to separate fully-, partially-,and un-conjugated species.

The linking domains can also have one or more polyalkylene glycolmoieties attached thereto. Such PAGs can be of varying lengths and maybe used in appropriate combinations to achieve the desired molecularweight of the composition.

The effect of a particular linker can be influenced by both its chemicalcomposition and length. A linker that is too long, too short, or formsunfavorable steric and/or ionic interactions with the target willpreclude the formation of complex between aptamer and target. A linker,which is longer than necessary to span the distance between nucleicacids, may reduce binding stability by diminishing the effectiveconcentration of the ligand. Thus, it is often necessary to optimizelinker compositions and lengths in order to maximize the affinity of anaptamer to a target.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

Example 1 Anti-C5 Aptamer Activity in the Classical and AlternativeComplement Pathways Example 1A: Hemolysis Assay

The CH50 test measures the ability of the complement system in a serumtest sample to lyse 50% of cells in a standardized suspension ofantibody-coated sheep erythrocytes. A solution of 0.2% human serum wasmixed with antibody-coated sheep erythrocytes (Diamedix EZ ComplementCH50 Kit, Diamedix Corp., Miami, Fla.) in the presence or absence ofvarious anti-C5 aptamers. The assay was run according to the kitprotocol in veronal-buffered saline containing calcium, magnesium and 1%gelatin (GVB⁺⁺ complement buffer) and incubated for 30 minutes at 37° C.After incubation, the samples were centrifuged to pellet intacterythrocytes. The optical density at 412 nm (OD412) of the supernatantwas read to quantify the release of soluble hemoglobin, which isproportional to the extent of hemolysis (Green et al., (1995) Chem.Biol. 2:683-95). To verify that the aptamers blocked C5 activation, somehemolysis supernatants were analyzed for the presence of C5a and C5b-9by ELISA (C5b-9 ELISA kit, Quidel, San Diego, Calif.; C5a ELISA kit, BDBiosciences, San Diego, Calif.) following the ELISA kit protocols.

The addition of a non-PEGylated anti-C5 aptamer (ARC186) (SEQ ID NO: 4)to the reaction mixture inhibited hemolysis in a dose-dependent manner,as shown in the graph of FIG. 7A, with an IC₅₀ of 0.5±0.1 nM, (see FIG.7B), a value that is consistent with the K_(D) determined bynitrocellulose filtration. At very high aptamer concentrations (>10 nM),the extent of hemolysis was essentially indistinguishable frombackground (no serum added), indicating that ARC186 (SEQ ID NO: 4) wasable to completely block complement activity. Conjugation of the ARC186(SEQ ID NO: 4) aptamer with 20 kDa (ARC657; SEQ ID NO: 61), 30 kDa(ARC658; SEQ ID NO: 62), branched 40 kDa (1,3-bis(mPEG-[20kDa])-propyl-2-(4′-butamide) (ARC187; SEQ ID NO: 5), branched 40 kDa(2,3-bis(mPEG-[20 kDa])-propyl-1-carbamoyl) (ARC1905; SEQ ID NO: 67),linear 40 kDa (ARC1537; SEQ ID NO: 65), and linear 2×20 kDa (ARC1730;SEQ ID NO: 66) PEG groups had little effect on the aptamer inhibitoryactivity in the CH50 hemolysis assay (FIG. 7A-FIG. 7D).

In an additional study, the inhibitory activity of the PEGylated anti-C5aptamer ARC1905 (branched 40 kDa (2,3-bis(mPEG-[20kDa])-propyl-1-carbamoyl); SEQ ID NO: 67) was compared to itsnon-PEGylated precursor, ARC672 (SEQ ID NO 63) which contains a terminal5′-amine, in the CH50 hemolysis assay described above. A solution ofhuman serum (Innovative Research, Southfield, Mich.) was mixed withantibody-coated sheep erythrocytes (Diamedix EZ Complement CH50 Kit,Diamedix Corp., Miami, Fla.) in the presence or absence of variousconcentrations of ARC1905 and ARC627 respectively such that the finalconcentration of serum in each assay was 0.1%, and the assay was runaccording to manufacturer's recommended protocol. The hemolysisreactions were incubated for 1 hour at 37° C. with agitation to ensurethat cells remained in suspension. At the end of the incubation, intactcells were pelleted by centrifugation (2000 rpm, 2 min, roomtemperature), 200 μL supernatant was transferred to a flat-bottomedpolystyrene plate (VWR, cat #62409-003). The optical density at 415 nm(OD415) of the supernatant was read to quantify the release of solublehemoglobin. The % inhibition at each aptamer concentration measured wascalculated using the equation %inh=100−100×(A_(sample)−A_(no serum))/(A_(no aptamer)−A_(no serum)),where A_(sample) is the sample absorbance at varying concentrations ofaptamer, A_(no serum) is the absorbance due to background hemolysis inthe absence of serum (100% inhibition control) and A_(no aptamer) is theabsorbance due to basal complement activity in the absence of aptamer(0% inhibition control). IC₅₀ values were determined from a plot of %inhibition versus [inhibitor] using the equation % inh=(%inh)_(maximum)×[inhibitor]^(n)/(IC₅₀+[inhibitor]”)+background. IC₉₀ andIC₉₉ values were calculated from IC₅₀ values using the equationsIC₉₀=IC₅₀×[90/(100−90]^(1/n) and IC₉₀=IC₅₀×[99/(100−99]^(1/n). The IC₅₀values for ARC1905 and ARC627 in this parallel study were 0.648+/−0.0521and 0.913+/−0.0679 respectively (see also FIG. 58) further confirmingthat PEGylation had little, if any, effect on aptamer function.

ELISA analysis of hemolysis supernatants indicated that this functionalinhibition correlated with blockade of C5a release. Thus, the hemolysisdata show that ARC186 (SEQ ID NO: 4), and its PEGylated conjugates, arehighly potent complement inhibitors that function by blocking theconvertase-catalyzed activation of C5.

Hemolysis assays with non-PEGylated material indicated that the anti-C5aptamer does not cross-react with C5 from a number of non-primatespecies, including rat, guinea pig, dog and pig. However, significantinhibitory activity was observed in screens of primate serum, includingserum from cynomolgus macaque, rhesus macaque and chimpanzee. The invitro efficacy of the anti-C5 aptamer was further investigated incynomolgus serum using ARC658 (SEQ ID NO: 62), the 30 kDa-PEG analogueof ARC186 (SEQ ID NO: 4). In a side-by-side comparison (n=3), ARC658inhibited human complement activity with an IC₅₀ of 0.21±0.0 nM andcynomolgus complement activity with an IC₅₀ of 1.7±0.4 nM (FIG. 8). ThusARC658 (SEQ ID NO: 62) is 8±3 fold less potent in cynomolgus serumcompared to human by this measure.

In a related study, the effects of the branched 40 kDa (2,3-bis(mPEG-[20kDa])-propyl-1-carbamoyl) PEGylated anti-C5 aptamer, ARC1905 (SEQ ID NO:67) on classical complement pathway activation as assayed by sheeperythrocyte hemolysis was investigated in the presence of human(Innovative Research, Southfield, Mich.), cynomolgus monkey(Bioreclamation, Hicksville, N.Y.), or rat serum (Bioreclamation,Hicksville, N.Y.). These assays were performed in highly diluted serum,0.1% for human and cynomolgus monkey, and 0.3% for rat, under the sameconditions as those used to compare the inhibitory effects of ARC1905against ARC672 on sheep erythrocyte hemolysis as described directlyabove. In a side by side comparison, complete inhibition (90-99%) of invitro complement activity was achievable with ARC1905 in both human andcynomolgus monkey sera whereas ARC1905 displayed little to no specificinhibitory activity in the rat complement sample (FIG. 59A). Similar toARC658, ARC1905 was ˜10-fold less potent against cynomolgus complementactivity under the conditions of the assay, as reflected in the IC₉₀ andIC₉₉ values reported in FIG. 59B.

Nitrocellulose Filter Binding Assays. Individual aptamers were³²P-labeled at the 5′ end by incubation with γ-³²P-ATP andpolynucleotide kinase (New England Biolabs, Beverly, Mass.).Radiolabeled aptamer was purified away from free ATP by gel-filtrationfollowed by polyacrylamide gel electrophoresis. To measure anti-C5aptamer affinity, radiolabeled aptamer (≤10 μM) was incubated withincreasing concentrations (0.05-100 nM) of purified C5 protein (Quidel,San Diego, Calif.) in phosphate-buffered saline containing 1 mM MgCl₂ atroom temperature (23° C.) and 37° C., for 15 min and 4 hr timeintervals. The binding reactions were analyzed by nitrocellulosefiltration using a Minifold I dot-blot, 96-well vacuum filtrationmanifold (Schleicher & Schuell, Keene, N.H.). A three-layer filtrationmedium was used, consisting (from top to bottom) of Protrannitrocellolose (Schleicher & Schuell), Hybond-P nylon (AmershamBiosciences, Piscataway, N.J.) and GB002 gel blot paper (Schleicher &Schuell). The nitrocellulose layer, which selectively binds protein overnucleic acid, preferentially retained the anti-C5 aptamer in complexwith a protein ligand, while non-complexed anti-C5 aptamer passedthrough the nitrocellulose and adhered to the nylon. The gel blot paperwas included simply as a supporting medium for the other filters.Following filtration, the filter layers were separated, dried andexposed on a phosphor screen (Amersham Biosciences) and quantified usinga Storm 860 Phosphorimager© blot imaging system (Amersham Biosciences).

As shown in shown in FIG. 9 and FIG. 10, increasing C5 concentrationsenhance the proportion of ARC186 captured on the nitrocellulosemembrane. The dependence of bound ARC186 on increasing C5 concentrationsis well-described by a single-site binding model (C5+ARC186↔C5⋅ARC186; %bound=C_(max)/(1+K_(D)/[C5]); C_(max) is the maximum % bound atsaturating [C5]; K_(D) is the dissociation constant). ARC186 bindingcurves at two temperatures following either a 15 min or a 4 hrincubation are shown in FIG. 9 and FIG. 10, respectively. Following a 15min incubation, the ARC186 binding curves at 23 and 37° C. areessentially indistinguishable within error, fitting with K_(D) values of0.5-0.6 nM (FIG. 9). Differences between binding curves at 23 and 37° C.become more pronounced when the incubation time is extended. Following a4 hr incubation (FIG. 10), the K_(D) observed at 23° C. decreases to0.08 0.01 nM, while the K_(D) observed at 37° C. remains unchanged(0.6±.0.1 nM).

To demonstrate the basis for the long incubation requirement at roomtemperature, the affinity at this temperature was further explored usingkinetic methods. The rate of the reverse reaction describing thedissociation of C5⋅ARC186 is v_(rev)=k⁻¹[C5⋅ARC186], where v_(rev) isthe rate (units of M min⁻¹) and k⁻¹ is the first order dissociation rateconstant (units of min⁻¹). The rate of the forward reaction describingthe formation of the C5⋅ARC186 complex is v_(for)=k₁[C5][ARC186], wherev_(for) is the rate (units of M min⁻¹) and k₁ is the second orderassociation rate constant (units of M⁻¹min⁻¹). The data were analyzedusing the pseudo-first order assumption, where the concentration of onereactant (C5 in this case) is held in vast excess over the other([C5]>>[ARC186], and thus remains essentially unchanged over the courseof the reaction. Under these conditions, the forward reaction isdescribed by the rate equation for a first order process,v_(for)=k₁′[ARC186], where k₁′=k₁[C5].

To analyze dissociation of C5⋅ARC186, radiolabeled ARC186 (≤10 μM) waspre-equilibrated with 5 nM C5 protein in phosphate-buffered salinecontaining 1 mM MgCl₂ at room temperature (23° C.). The dissociationreaction was initiated by the addition of non-labeled ARC186 (1 μM),which acts as a trap for free C5, and stopped by nitrocellulosefiltration partitioning of bound and free radiolabeled ARC186. Atimecourse of ARC186 dissociation was obtained by varying the durationbetween initiation of the dissociation reaction and filtration. Thetimecourse of dissociation, observed as a decrease in the percentage ofradiolabeled ARC186 captured on the nitrocellulose filter (equal to thepercent bound to C5), is well-described by a single-exponential decaywhere % ARC186 bound=100×e^(−k) ⁻¹ ^(t) (see FIG. 11). The value of thedissociation rate constant, k⁻¹, determined by this method is 0.013±0.02min⁻¹, corresponding to a half-life (t_(1/2)=ln 2/k⁻¹) of 53±8 min.

To analyze the association reaction, the equilibration rate constant(k_(eq)) for the formation of C5⋅ARC186 was measured in the presence ofvarying concentrations of C5 protein (1-5 nM). Complex formation wasinitiated by mixing together C5 protein and radiolabeled ARC186 in PBScontaining 1 mM MgCl₂ at room temperature (23° C.), and stopped bynitrocellulose filtration partitioning. As described for thedissociation reactions, a timecourse of complex formation was obtainedby varying the duration between the initiation of the reaction andfiltration. The timecourse of equilibration, observed as an increase inthe percentage of radiolabeled ARC186 captured on the nitrocellulosefilter, is well described by a single-exponential decay where % ARC186bound=100×(1−e^(−k) ⁻¹ ^(t)). The timecourses of equilibration for 1, 2and 4 nM C5 are displayed in FIG. 12. As expected, the value of k_(eq)increases linearly with [C5] (k_(eq) (1 nM)=0.19±0.02 min⁻¹; k_(eq) (2nM)=0.39±0.03 min⁻¹; k_(eq) (3 nM)=0.59±0.05 min⁻¹; k_(eq) (4nM)=0.77±0.06 min⁻¹; k_(eq) (5 nM)=0.88±0.06 min⁻¹). Under theconditions of the experiment, the relationship between k_(eq), k₁ andk⁻¹ is k_(eq)=k₁[C5]+k⁻¹. Thus, an estimate of k₁ is derived from theslope of a plot of k_(eq) versus [C5] (see FIG. 12 inset), in this case0.18±0.01 nM⁻¹ min⁻¹.

These data indicate that, under conditions of low C5 concentration(e.g., 0.1 nM), an extended incubation is required in order for themixture of C5 and radiolabeled. ARC186 to reach equilibrium. Under theseconditions, k_(eq)=(0.18±0.01 nM⁻¹ min⁻¹) (0.1 nM)+0.013 min⁻¹=0.03min⁻¹, corresponding to a half-life of 22 min. Thus, nearly 2 hours ofroom temperature incubation (˜5 half-lives) are required for complete(>95%) equilibration. A short incubation time (e.g., 15 min) willsignificantly underestimate the actual affinity of the complex, as shownabove by the difference in affinities observed for a 15 min (K_(D)=0.5nM) versus a 4 hour (K_(D)=0.08 nM) incubation. An alternative estimateof the room temperature K_(D) can be calculated from the kinetic dataaccording to the relationship K_(D)=k⁻¹/k₁. In this case, the calculatedK_(D) is 0.07±0.01 nM, which is completely consistent with the K_(D)determined above by thermodynamic methods.

The specificity of ARC186 (SEQ ID NO: 4) for C5 was also assessed innitrocellulose filtration assays by comparison with complementcomponents both upstream and downstream from C5 in the complementcascade. Purified human proteins and protein complexes were purchasedfrom Complement Technologies (Tyler, Tex.) including: C1q (cat.#A099.18; 2.3 μM), C3 (cat. #A113c.8; 27 μM), C5 (cat. #A120.14; 5.4μM), C5a des Arg (cat. #A145.6; 60 μM), sC5b-9 (cat. #A127.6; 1 μM),factor B (cat. #A135.12; 11 μM) and factor H (cat. #A137.13P; 6.8 μM).Binding reactions were established by performing serial dilutions ofprotein in PBS plus 1 mM MgCl₂, 0.02 mg/mL BSA and 0.002 mg/mL tRNA,incubating for 1-4 hours at 25° C. or 37° C., and then applied to thenitrocellulose filtration apparatus as described above. Dissociationconstants K_(D) were determined from semi-log plots of % nitrocellulosebinding versus [C5] by a fit of the data to the equation: %nitrocellulose binding=amplitude×[C5]/(K_(D)+[C5]).

The results depicted in FIG. 13 show the aptamer essentially does notrecognize C5a (K_(D)>>3 μM), although it does display weak affinity forsoluble C5b-9 (K_(D)>0.2 μM), presumably due to interactions with theC5b component. Other complement components display moderate to weakaffinity for the aptamer. Non-activated C3 essentially does not bind tothe aptamer; however, factor H (K_(D)˜100 nM) and, to a much lesserextent, C1q (K_(D)>0.3 μM) do bind. Taken together, the data indicatethat ARC186 (SEQ ID NO: 4) binds with high affinity to human C5, mainlyvia recognition of the C5b domain. Thus, ARC186 and its PEGylatedderivatives e.g., ARC1905 should not interfere with generation of C3b,which is important for bacterial opsonization, or with innate control ofC′ activation by regulatory factors.

Conjugation of aptamers with high molecular weight PEG moietiesintroduces the possibility of steric hindrance leading to reducedaffinity. PEG-modified aptamers are not readily evaluated for directbinding by nitrocellulose filtration assays due to the tendency of theseaptamers to adhere to nitrocellulose even in the absence of targetprotein. However, the relative affinities of these aptamers can beassessed from their comparative ability to compete with radiolabeled,non-PEGylated aptamer (≤10 μM) for binding to target as measured bynitrocellulose filtration known as a competition binding assay, run at37° C. As the concentration of cold (i.e., non-radiolabeled) competitorincreases, the percent of radiolabeled aptamer bound to target proteindecreases. As shown in FIG. 14, increasing concentrations of cold ARC186(SEQ ID NO: 4) or PEGylated aptamer (ARC657 (SEQ ID NO: 61), ARC658 (SEQID NO: 62), and ARC187 (SEQ ID NO: 5)) (0.05-1000 nM) readily competewith radiolabeled ARC186 (SEQ ID NO: 4) for binding in the presence of 2nM C5 protein. Additionally, the titration curves for all four aptamersnearly overlap, indicating that PEG-conjugation in the case of ARC657,ARC658 and ARC 187 has little or no effect on the affinity of theaptamer for C5.

In a similar study, the effect of PEG conjugation on binding to C5 wastested by comparing ARC672 (ARC186 with a 5′-terminal amine; SEQ ID NO63) with ARC1905 (ARC627 conjugated with a branched 40 kDa(2,3-bis(mPEG-[20 kDa])-propyl-1-carbamoyl) PEG) using the competitionbinding assay. 10 μM stocks of each aptamer were prepared in PBS plus 1mM MgCl₂, 0.01 mg/mL BSA, 0.002 mg/mL tRNA, and serially diluted togenerate a 10× sample series covering a >100-fold range of aptamerconcentration. 12 μL aliquots of each sample were then added in a96-well plate to 96 μL ³²P-radiolabeled ARC186 to generate a 1.1×solution of label and cold competitor. 90 μL of label/competitorsolution was then added to 10 μL of 10× C5 protein to initiate thereactions. The final concentration of radiolabeled ARC186 in eachreaction was held constant. Binding reactions were equilibrated for15-30 min at 37° C., and then filtered onto nitrocellulose filterapparatus described above. For the purposes of data analysis, coldcompetitor aptamers were treated as competitive inhibitors of theARC186/C5 interaction; % inhibition was calculated by normalizing thedata to control reactions lacking competitor (0% inhibition control).IC₅₀ values were determined from semi-log plots of % inhibition versus[ARC672] or [ARC1905] by a fit of the data to the equation: %inhibition=amplitude×[competitor]^(n)/(IC₅₀ ^(n)+[competitor]^(n)).

As shown in FIG. 60, the addition of a branched 40 kDa (2,3-bis(mPEG-[20kDa])-propyl-1-carbamoyl) PEG had little or no effect on aptameraffinity as measured by competitive binding. K_(D) values of0.46+/−0.149 nM and 0.71+/−0.130 nM were approximated for ARC672 andARC1905 respectively by the y-intercept of the line fit to the IC₅₀versus C5 data in FIG. 60. Both values are close to the K_(D) determinedfor ARC186 at 37° C.

The temperature dependence of the interaction between ARC1905 and C5 wasalso estimated by competition assay. ARC1905 was serially diluted togenerate 10× sample series as described above. Binding reactions wereequilibrated for 1-4 hours at 25° C. or 37° C., and then filtered ontothe nitrocellulose filter apparatus. Percent inhibition was calculatedby normalizing the data to control reactions lacking competitor (0%inhibition control) or lacking C5 protein (100% inhibition control).IC₅₀ values were determined from semi-log plots of % inhibition versus[ARC672] or [ARC1905] by a fit of the data to the equation: %inhibition=amplitude×[competitor]^(n)/(IC₅₀ ^(n)+[competitor]^(n)). Asshown in FIG. 61 ARC1905 binds to C5 with high affinity at both 25° C.and 37° C. K_(D) values of 0.15±0.048 nM and 0.69±0.148 nM were obtainedat 25° C. and 37° C., respectively, from the y-intercept of the IC₅₀versus C5 data. Both values are consistent with the K_(D) valuesdetermined for the ARC186/C5 interaction described above.

Example 1B: Whole Blood Assay

The effect of the anti-C5 aptamer on the alternative pathway of thecomplement system was analyzed using the following whole blood assay. Inthe absence of an anticoagulant, blood was drawn from normal humanvolunteers. Aliquots of blood (containing no anti-coagulant) wereincubated with increasing concentrations of ARC186 (SEQ ID NO: 4) for 5hours at room temperature or 37° C. Samples were centrifuged to isolateserum and the presence of C5b in the serum was detected by sC5b-9 ELISA(C5b-9 ELISA kit, Quidel, San Diego, Calif.). As shown in FIG. 15, theanti-complement activity, as reflected in production of C5b-9, betweensamples incubated at different temperatures diverged at 3 μM. The roomtemperature data indicated that the concentration of aptamer requiredfor quantitative inhibition is in the range of 3-6 μM, whereas thereported concentration of C5 is approximately 400 nM. These resultssuggest that greater than 10-fold molar excess of anti-C5 aptamer(ARC186; SEQ ID NO: 4) may be required for complete inhibition of C5activity.

Example 1C: Complement Activation by Zymosan

Zymosan is a polysaccharide component of the yeast cell wall, and apotent activator of the alternative complement cascade. Addition ofzymosan to ex vivo samples of blood, plasma or serum results in theaccumulation of complement activation products, including C5a and thesoluble version of C5b-9 (sC5b-9). Samples of undiluted human serum(Center for Blood Research, Boston, Mass.), citrated human whole blood(Center for Blood Research, Boston, Mass.) or cynomolgus serum (CharlesRiver Labs, Wilmington, Mass.) were spiked with increasingconcentrations of ARC658 (SEQ ID NO: 62), the 30K PEG analog of ARC186(SEQ ID NO: 4). To activate complement, zymosan (Sigma, St. Louis, Mo.)in a 10× suspension was added to samples to a final concentration of 5mg/mL. Following a 15 minute incubation at 37° C., zymosan particleswere removed by centrifugation and the extent of complement activationwas determined by C5a and/or sC5b-9 ELISA (C5b-9 ELISA kit, Quidel, SanDiego, Calif.; C5a ELISA kit, BD Biosciences, San Diego, Calif.).

In the absence of aptamer, zymosan treatment activates ˜50% of serum orwhole blood C5, compared to ˜1% activation in untreated sample. Additionof anti-C5 aptamer up to 50 nM (˜10% of C5 concentration in blood) hadlittle effect on sC5b-9 formation. However, further titration of C5 withincreasing concentrations of ARC658 (SEQ ID NO: 62) inhibited C5activation in a dose-dependent manner as seen in FIG. 16. In human serumor whole blood, quantitative (˜99%) inhibition was observed at 0.8-1 μMARC658 (SEQ ID NO: 62), corresponding to ˜2 molar equivalents of aptamerto C5. Higher concentrations of aptamer were required to achievecomparable inhibition in cynomolgus serum. In this case, 99% inhibitionwas achieved only in the presence of 10 μM aptamer, or ˜20 molarequivalents of aptamer to C5.

In a similar study, the inhibitory effects of ARC1905 (the branched 40kDa (2,3-bis(mPEG-[20 kDa])-propyl-1-carbamoyl) PEGylated version ofARC186) was tested on human and cynomolgus monkey samples using thezymosan to activate complement via the alternative pathway as follows.Zymosan A from Saccharomyces cerevisiae was supplied by Sigma-Aldrich,Inc. (cat. no. Z4250-1G, St. Louis, Mo.). The zymosan A was supplied asa powder and was resuspended in Dulbecco's PBS (Gibco, Carlsbad, Calif.,cat. no. 14190-144) to yield a 50 mg/mL suspension. Frozen, poolednormal human serum (cat. no. IPLA-SER) was purchased from InnovativeResearch (Southfield, Mich.). Frozen, pooled cynomolgus macaque serum(cat. no. CYNSRM) was purchased from Bioreclamation (Hicksville, N.Y.).Vials of 5-10 mL serum provided by the supplier were thawed at 37° C.,aliquoted (˜1 mL) and stored at −20° C. Aliquots were thawed as neededjust prior to use by incubation at 37° C. and stored on ice duringexperiments. The final concentration of serum in each assay was ˜100%. A20 μM stock of ARC1905 was prepared in 0.9% saline and serially dilutedto generate a 10× sample series covering a ˜90-fold range of aptamerconcentrations. A no-aptamer (saline only) sample was also included as anegative (0% inhibition) control.

90 μL of serum was pipetted into wells of a 96-well PCR plate (VWR, cat.no. 1442-9596). 10 μL of aptamer sample was diluted directly into theserum at room temperature and mixed. 8 μL of 50 mg/mL zymosan waspipetted into wells of a separate 96-well PCR plate. Both plates weresimultaneously pre-incubated at 37° C. for 15 minutes. Immediatelyfollowing the pre-incubation, 80 μL of the serum/aptamer mixture wasadded directly to 8 μL of zymosan and mixed, yielding 5 mg/mL zymosanfinal concentration. The reaction plate was sealed and incubated for 15minutes at 37° C. At the end of the incubation, the reaction wasquenched by pipetting 8 μL 0.5M EDTA into the wells and mixing. Thezymosan was pelleted by centrifugation (3700 rpm, 5 min, roomtemperature) and ˜80 μL quenched supernatant was transferred to a new96-well PCR plate and sealed. Supernatants were flash frozen in liquidnitrogen and stored at −20° C. To control for zymosan-independentbackground activation, serum samples were prepared and treated exactlyas described above, except that 8 μL of saline was added instead ofzymosan.

The extent of C5 activation was determined from the relative levels ofC5a generated in each zymosan-activated sample, as measured by C5a ELISA(ALPCO Diagnostics, Windham, N.H., cat. no. EIA-3327) following the C5aELISA kit protocol. The C5a ELISA kit includes human specific reagentsand is formatted for analysis of human C5a (hC5a) in plasma or serumsamples. It was therefore necessary to characterize the response of theELISA to cynomolgus monkey C5a using cynomolgus concentration standards.To prepare a set of custom standards, 0.5 mL aliquots of human orcynomolgus monkey serum were incubated with 5 mg/mL zymosan for 15 minat 37° C., quenched with 12.5 μL 0.5M EDTA and centrifuged to remove thezymosan. The concentration of C5a in the zymosan-activated human serumsample was determined to be approximately 2 μg/mL hC5a by comparison tohC5a standard plasmas provided with the kit. The concentration of C5a inthe cynomolgus monkey sample, expressed in human C5a equivalents (hC5aeq), was determined to be approximately 0.6 μg/mL hC5a eq. Series ofstandards covering a range from 0.4-400 ng/mL hC5a or 0.12-120 ng/mLhC5a eq were prepared by dilution into rat serum (which does notinterfere with the ELISA). Standards were pre-treated with aprotein-precipitating reagent as directed in the ELISA kit protocol andapplied without further dilution to the ELISA plate. The ELISA plate wasread at an aborbance maximum of 450 nm (A₄₅₀) using a VersaMax UV/visabsorbance plate reader (Molecular Dynamics, Sunnyvale, Calif.). TheA₄₅₀ varied with C5a concentration from a low of 0.1-0.2 at low C5a,plateauing ˜3.5 at high C5a. For the purposes of quantifying C5a inassay samples, the upper and lower limits of quantification were,respectively, 25 and 0.78 ng/mL hC5a for human, and 15 and 0.94 ng/mLhC5a eq for cynomolgus monkey. A₄₅₀ versus ng/mL hC5a or hC5a eq wasplotted as shown in FIG. 62, and a standard curve was obtained from a4-parameter fit to the data using the equation y=((A−D)/(1(x/C)^(B)))+D.

Just prior to C5a analysis, assay samples (including the saline-only andno-zymosan controls) were pre-treated with protein-precipitating reagentas directed in the ELISA kit protocol, then serially diluted in 0.9%saline. C5a levels in undiluted assay samples (including some of theno-zymosan controls) typically exceeded the upper limit of quantitation(ULOQ). Therefore, dilutions of 1/5, 1/50 and 1/250 were tested toaccommodate the full range of assay sample C5a concentrations. C5alevels were quantified by comparison with the appropriate (human orcynomolgus monkey) standard curve and corrected for dilution. The %inhibition at each aptamer concentration was calculated using theequation %inh.=100−100×(C5a_(sample)−C5a_(no-zymosan))/(C5a_(saline-only)−C5a_(no-zymosan)).IC₅₀ values were determined from a plot of % inhibition versus [AR01905]using the equation % inh=(% inh.)_(maximum)×[ARC1905]^(n)/(IC₅₀^(n)+[ARC1905]^(n)) background. IC₉₀ and IC₉₉ values were calculatedfrom IC₅₀ values using the equations IC₉₀=IC₅₀×[90/(100−90]^(1/n) andIC₉₉=IC₅₀×[99/(100−99]^(1/n).

The extent of C3 activation (the step in the common complement pathwayjust upstream of C5) was determined from the relative levels of C3agenerated in each zymosan-activated sample, as measured by C3a ELISA(Becton-Dickinson OptiEIA C3a ELISA kit, cat. no. 550499, FranklinLakes, N.J.) following the C3a ELISA kit protocol.

Just prior to C3a analysis, samples (including the saline-only andno-zymosan controls) were serially diluted in 0.9% saline. The C3a ELISAis more sensitive than that for C5a; therefore, dilutions of 1/500,1/5000 and 1/25,000 were necessary to accommodate the full range ofsample C3a concentrations. Kit standards, derived from human serum, wereused instead of the custom standards prepared for C5a analysis. SinceC3a levels did not vary greatly, the human-specific standards provided asufficient indication of their relative levels.

The data generated from both the C5a and C3 ELISAs were analyzed usingMicrosoft Excel, and the mean % inhibition values were plotted usingKaleidagraph (v. 3.51, Synergy Software). IC₅₀, IC₉₀ and IC₉₉ valueswere determined using the XLfit 4.1 plug-in to Excel. The comparativeeffects of ARC1905 on human and cynomolgus monkey complement activation,as measured by this approach, are summarized in FIG. 63 and FIG. 64. Ascan be seen from these Figs., complete inhibition of C5 activation viathe alternate pathway is achievable in vitro with ARC1905 in both humanand cynomolgus monkey sera. In human serum, the concentration of ARC1905required for 90% inhibition of C5 activation in an undiluted sample was442±23 nM, approximately equivalent to the average molar concentrationof C5. However, ARC1905 was 4-6-fold less potent against cynomolgusmonkey complement activity under the conditions of the assay, asreflected in the IC₉₀ and IC₉₉ values.

The effects of ARC1905 C3 activation, as measured by C3a levels, aresummarized in FIG. 65. The rationale for specifically targeting the tailend of the complement pathway is to block the pro-inflammatory functionsof C5a and the membrane attack complex (MAC) without compromising thepathogen-fighting functions of upstream factors culminating in C3a andC3b generation. The data in FIG. 65 demonstrates that ARC1905, up to 2μm, does not inhibit C3a generation and indicates that upstreamcomplement activation is not negatively impacted by ARC1905. Essentiallycomplete blockade of alternate pathway C5 activation was achieved inboth human and cynomolgus monkey serum samples using ARC1905. ARC1905was approximately an order of magnitude less potent in inhibitingcynomolgus monkey C5 activation than human C5 activation under theconditions of this assay. While not wishing to be bound by theory, theinhibitory effect of ARC1905 on complement activation is specific to C5since activation of C3 was not inhibited.

Example 1D: Tubing Loop Model of Complement Activation

To test the ability of anti-C5 aptamer to block complement activationinduced by exposure to foreign materials, as found in a cardiopulmonarybypass circuit, we used the tubing loop model described by Nilsson andcolleagues (Gong et al, (1996) Journal of Clinical Immunology 16, 222-9;Nilsson et al, (1998) Blood 92, 1661-7). Tygon S-50-HL medical/surgicaltubing (¼″ inner diameter) (United States Plastic Corp. ((Lima, Ohio),cat. #00542) was cut into lengths of approximately 300 mm (approximately9 mL volume) and filled with 5 mL human donor blood containing 0.4units/mL heparin (Celsus) and varying concentrations of ARC658 (SEQ IDNO: 62) (0-5 μM). Each length of Tygon tubing was closed into a loopwith short sections (˜50 mm) of non-surgical silicone linker tubing (⅜″inner diameter) (United States Plastic Corp. (formulation R-3603, cat.#00271) as described in Gong et al. Tubing loops were rotated for 1 hourat approximately 30 rpm in a 37° C. water bath. The loop contents werethen poured into polypropylene conical tubes containing EDTA (10 mMfinal concentration) to quench complement activation. Platelet-poorplasma was isolated by centrifugation and analyzed for C5a and C3a byELISA (C3a ELISA kit, Quidel, San Diego, Calif.; C5a ELISA kit, BDBiosciences, San Diego, Calif.).

The total complement activation in the absence of aptamer was smallcompared to the zymosan assay. Typically, C5a levels increased byapproximately 6 ng/mL following the 1 hour incubation, corresponding toactivation of <1% of the available C5. Nevertheless, this increase wasreproducible and inhibited by titration with ARC658 (SEQ ID NO: 62). Asshown in FIG. 17, 300-400 nM ARC658 (SEQ ID NO: 62) was sufficient toachieve 99% inhibition of C5 activation, a level that is approximatelyequivalent or slightly less than the molar concentration of C5 in blood.While not wishing to be bound by any theory, although less aptamer isrequired to obtain 99% inhibition of C5 activation in this model than inthe zymosan model, this observation could reflect the substantialdifferences in the complement-activating stimulus used in the twoassays. C3a generation was also monitored as a control to verify thatARC658 (SEQ ID NO: 62) did not block activation steps earlier than C5 inthe complement cascade. C3a levels increased by approximately 4000 ng/mLfollowing the 1 hour incubation, corresponding to activation of around10% of the available C3. In contrast to C5a generation, little dosedependent inhibition of C3a generation was observed upon titration withARC658 (SEQ ID NO: 62) demonstrating that ARC658 (SEQ ID NO: 62)specifically blocks C5 cleavage.

The tubing loop model study was repeated with the anti-C5 aptamerARC1905 (SEQ ID NO 67). ARC1905 was serially diluted in 0.9% saline togenerate a 20× sample series covering a 100-fold range of aptamerconcentrations (10-1000 nM final in the assay). Samples containingirrelevant aptamer (ARC127) were included to control for non-specificoligonucleotide effects. A no-aptamer (saline only) sample was alsoincluded as a negative controlSingle-donor blood samples were drawn bystandard phlebotomy methods from in-house volunteers. Whole blood wasdrawn from 5 separate donors directly into a 60 mL syringe(Becton-Dickinson, (Franklin Lakes, N.J.), cat. #309653) and immediatelyaliquoted into bivalirudin (20 μM final) (Prospec-Tany Technogene Ltd.,(Israel), lot #105BIV01)+/−aptamer. The anti-coagulant bivalirudin, adirect thrombin inhibitor, was used instead of heparin which interfereswith complement activation.

The tubing loop model was performed essentially as described immediatelyabove. ˜300 mm sections of tube (diameter ¼″, volume ˜9 mL) were filledwith 5 mL of blood/aptamer/bivalirudin samples immediately after theblood had been drawn from the donor. The tubes were then securelyfastened into loops with short sections (˜50 mm) of silicone linkertubing, yielding a gas volume of ˜4 mL. The tubing loops were rotatedvertically at 32 rpm during incubation in a 37° C. water bath for 1hour. After incubation, all 5 mL of sample was transferred to a 15 mLconical tube (Corning, (Corning, N.Y.), cat. #430766) containing 100 μLof 0.5M EDTA, giving a final EDTA concentration of 10 mM. 1 mL of plasmasupernatant was collected from each quenched sample followingcentrifugation (Eppendorf Centrifuge 5804) at 4° C. (3,300 rpm, 20minutes). Supernatants were flash frozen in liquid nitrogen and storedat −20° C. To control for background activation, a pre-CPB sample wasprepared by adding 5 mL of fresh blood directly to a 15 mL conical tubeon ice containing 100 μL 0.5M EDTA. This sample was processed for plasmaand stored as described above.

The extent of C5 activation was determined from the relative levels ofC5a generated in each activated sample, as measured by C5a ELISA asdescribed immediately above. The C5a ELISA was performed on undilutedplasma samples according the ELISA kit protocol and sample C5a levelswere quantified by comparison with the C5a standards provided by themanufacturer. The % inhibition of C5a generation at each aptamerconcentration was calculated using the equation %inh=100−100×(C5a_(sample)−C5a_(pre-CPB))(C5a_(saline-only)−C5a_(pre-CPB)).IC₅₀ values were determined from a plot of % inhibition versus [ARC1905]using the equation % inh=(% inh.)_(maximum)×[ARC1905]^(n)/(IC₅₀^(n)+[ARC1905]^(n))+background. IC₉₀ and IC₉₉ values were calculatedfrom IC₅₀ values using the equations IC₉₀=IC₅₀×[90/(100−90]^(1/n) andIC₉₉=IC₅₀×[99/(100−99]^(1/n).

The extent of C3 activation was determined from the relative levels ofC3a generated in each activated sample, as measured by C3a ELISA asdescribed immediately above. Just prior to C3a analysis, samples(including the saline-only and pre-CPB controls) were serially dilutedin 0.9% saline. The C3a ELISA is more sensitive than that for C5a;therefore, a dilution of 1/5000 was necessary to accommodate the rangeof sample C3a concentrations. Sample C3a levels were quantified bycomparison to kit standards, and % inhibition was calculated asdescribed for C5a. The data were analyzed using Microsoft Excel, and themean % inhibition values were plotted using Kaleidagraph (v3.5 SynergySoftware). IC₅₀, IC₉₀ and IC₉₉ values were determined using the XLfit4.1 plug-in to Excel.

The mean effects of ARC1905 and irrelevant aptamer, ARC127, oncomplement activation in the five donors is summarized in FIG. 66. Asshown in FIG. 67 complete blockade of C5 activation, as reflected in thegeneration of C5a, was achieved with <500 nM ARC1905, while theirrelevant aptamer had no inhibitory effect up to 1 μM. The mean wholeblood IC₅₀, IC₉₀ and IC₉₉ values were 119±28.6 nM, 268±39.2 nM and694±241 nM, respectively (FIG. 66). While not wishing to be bound bytheory, it is reasonable to assume that ARC1905 is excluded from thecellular blood volume, which comprises approximately 45% of the total.The IC₅₀, IC₉₀ and IC₉₉ values, adjusted to reflect C5 inhibition inplasma, therefore, were 216±52.0 nM, 487±71 nM and 1261±438 nM. Thesevalues are consistent with the parameters calculated for ARC1905inhibition of zymosan-induced complement activation in serum suggestingthat cellular blood components do not interfere significantly withARC1905 anti-C5 activity. C3a generation was not inhibited by ARC1905 orirrelevant aptamer up to 1 μm. While not wishing to be bound by theory,this suggests that ARC1905 neither inhibits the C3 convertase reaction,nor blocks other steps that contribute to alternate cascade activationsuch as C3 deposition and convertase assembly.

Example 2 De Novo Selections and Sequences

C5 Selection with dRmY Pool

Two selections were performed to identify dRmY aptamers to human fulllength C5 protein. The C5 protein (Quidel Corporation, San Diego, Calif.or Advanced Research Technologies, San Diego, Calif.) was used in fulllength (“FL”) and partially trypsinized (“TP”) form and both selectionswere direct selections against the protein targets which had beenimmobilized on a hydrophobic plate. Both selections yielded poolssignificantly enriched for full length C5 binding versus naïve,unselected pool. All sequences shown in this example are shown 5′ to 3′.

Pool Preparation: A DNA Template with the Sequence

TAATACGACTCACTATAGGGAGAGGAGAGAACGTTCTACN₍₃₀₎GGTCGATC GATCGATCATCGATG(ARC520; SEQ ID NO: 70) was synthesized using an ABI EXPEDITE™ DNAsynthesizer, and deprotected by standard methods. The templates wereamplified with 5′ primer TAATACGACTCACTATAGGGAGAGGAGAGAACGTTCTAC (SEQ IDNO: 71) and 3′ primer CATCGATGATCGATCGATCGACC (SEQ ID NO: 72) and thenused as a template for in vitro transcription with Y639F single mutantT7 RNA polymerase. Transcriptions were done using 200 mM HEPES, 40 mMDTT, 2 mM spermidine, 0.01% TritonX-100, 10% PEG-8000, 9.5 mM MgCl₂, 2.9mM MnCl₂, 2 mM NTPs, 2 mM GMP, 2 mM spermine, 0.01 units/μL inorganicpyrophosphatase, and Y639F single mutant T7 polymerase.

Selection: In round 1, a positive selection step was conducted onnitrocellulose filter binding columns. Briefly, 1×10¹⁵ molecules (0.5nmoles) of pool RNA were incubated in 100 μL binding buffer (1×DPBS)with 3 μM full length C5 or 2.6 μM partially trypsinized C5 for 1 hourat room temperature. RNA:protein complexes and free RNA molecules wereseparated using 0.45 um nitrocellulose spin columns from Schleicher &Schnell (Keene, N.H.). The columns were pre-washed with 1 mL 1×DPBS, andthen the RNA:protein containing solutions were added to the columns andspun in a centrifuge at 1500 g for 2 min. Three buffer washes of 1 mLwere performed to remove nonspecific binders from the filters, then theRNA:protein complexes attached to the filters were eluted twice with 200μl washes of elution buffer (7 M urea, 100 mM sodium acetate, 3 mM EDTA,pre-heated to 95° C.). The eluted RNA was precipitated (2 μL glycogen, 1volume isopropanol, 12 volume ethanol). The RNA was reverse transcribedwith the ThermoScript RT-PCR™ system (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions, using the 3′ primerdescribed above SEQ ID NO: 72, followed by PCR amplification (20 mM TrispH 8.4, 50 mM KCl, 2 mM MgCl₂, 0.5 uM primers SEQ ID NO: 71 and SEQ IDNO: 72, 0.5 mM each dNTP, 0.05 units/μL Taq polymerase (New EnglandBiolabs, Beverly, Mass.)). The PCR templates were purified usingCentricep columns (Princeton Separations, Princeton, N.J.) and used totranscribe the next round pool.

In subsequent rounds of selection, separation of bound and free RNA wasdone on Nunc Maxisorp hydrophobic plates (Nunc, Rochester, N.Y.). Theround was initiated by immobilizing 20 pmoles of both the full length C5and partially trypsinized C5 to the surface of the plate for 1 hour atroom temperature in 100 μL of 1×DPBS. The supernatant was then removedand the wells were washed 4 times with 120 μL wash buffer (1×DPBS). Theprotein wells were then blocked with a 1×DPBS buffer containing 0.1mg/mL yeast tRNA and 0.1 mg/mL salmon sperm DNA as competitors. The poolconcentration used was always at least in five fold excess of theprotein concentration. The pool RNA was also incubated for 1 hour atroom temperature in empty wells to remove any plastic binding sequences,and then incubated in a blocked well with no protein to remove anycompetitor binding sequences from the pool before the positive selectionstep. The pool RNA was then incubated for 1 hour at room temperature andthe RNA bound to the immobilized C5 was reverse transcribed directly inthe selection plate by the addition of RT mix (3′ primer, SEQ ID NO: 72and Thermoscript RT, Invitrogen) followed by incubation at 65° C. for 1hour. The resulting cDNA was used as a template for PCR (Taq polymerase,New England Biolabs). Amplified pool template DNA was desalted with aCentrisep column (Princeton Separations) according to the manufacturer'srecommended conditions and used to program transcription of the pool RNAfor the next round of selection. The transcribed pool was gel purifiedon a 10% polyacrylamide gel every round.

The selection progress was monitored using a sandwich filter binding(dot blot) assay. The 5′-³²P-labeled pool RNA (trace concentration) wasincubated with C5, 1×DPBS plus 0.1 mg/mL tRNA and 0.1 mg/mL, salmonsperm DNA, for 30 minutes at room temperature, and then applied to anitrocellulose and nylon filter sandwich in a dot blot apparatus(Schleicher and Schuell). The percentage of pool RNA bound to thenitrocellulose was calculated and monitored approximately every 3 roundswith a single point screen (+/−300 nM C5). Pool K_(d) measurements weremeasured using a titration of protein and the dot blot apparatus asdescribed above.

Selection data: Both selections were enriched after 10 rounds over thenaïve pool. See FIG. 18. At round 10, the pool K_(d) was approximately115 nM for the full length and 150 nM for the trypsinized selection, butthe extent of binding was only about 10% at the plateau in both. The RIOpools were cloned using TOPO TA cloning kit (Invitrogen) and sequenced.

Sequence Information: 45 clones from each pool were sequenced. RIO fulllength pool was dominated by one single clone ARC913 (SEQ ID NO: 75)which made up 24% of the pool, 2 sets of duplicates and single sequencesmade up the remainder. The RIO trypsinized pool contained 8 copies ofthe same sequence ARC913 (SEQ ID NO: 75), but the pool was dominated byanother sequence (AMX221.A7; 46%). The clone ARC913 (SEQ ID NO: 75) hada K_(d) about 140 nM and the extent of binding went to 20%. See FIG. 19.

The individual sequence listed in Table 5 is listed in the 5′ to 3′direction, and represents the ribonucleotide sequence of the aptamerthat was selected under the dRmY SELEX™ conditions provided. In theembodiments of the invention derived from this selection (and asreflected in the sequence listing) the purines (A and G) are deoxy andthe pyrimidines (U and C) are 2′-OMe. The sequence listed in Table 5 mayor may not contain capping (e.g., a 3′-inverted dT). The unique sequenceof the aptamer below begins at nucleotide 23, immediately following thefixed sequence GGGAGAGGAGAGAACGUUCUAC (SEQ ID NO: 73), and runs until itmeets the 3′fixed nucleic acid sequence GGUCGAUCGAUCGAUCAUCGAUG (SEQ IDNO: 74)

TABLE 5 Nucleotide sequence of the C5 dRmY aptamerARC913 (SEQ ID NO: 75)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUACAUACGCAGGGGUCGAUCGAUCGAUCAUCGAUG

Hemolysis Assay: The effect of ARC913 (SEQ ID NO: 75) on the classicalpathway of the complement system was analyzed using a hemolysis assaypreviously described, compared to both ARC186 (SEQ ID NO: 4) (Anti-C5aptamer, positive control) and unselected dRmY pool (negative control).In the assay of hemolytic inhibition, a solution of 0.2% whole humanserum was mixed with antibody-coated sheep erythrocytes (Diamedix EZComplement CH50 Test, Diamedix Corporation, Miami, Fla.) in the presenceof titrated ARC913 (SEQ ID NO: 75). The assay was run inveronal-buffered saline containing calcium, magnesium and 1% gelatin(GVB⁺⁺ complement buffer) and incubated for 1 hr at 25° C. Afterincubation the samples were centrifuged. The optical density at 415 run(OD₄₁₅) of the supernatant was read. The inhibition of hemolysisactivity is expressed as % hemolysis activity compared to control. SeeFIG. 20. The IC₅₀ of the aptamer was calculated to be about 30 nM.

Example 3 Composition and Sequence Optimization Example 3A: Minimizationof ARC913

Six constructs based on ARC913 (SEQ ID NO: 75) were transcribed, gelpurified, and tested in dot blots for binding to C5. ARC954 was similarto the parent clone with a K_(d) of 130 nM and extent of binding at 20%,while ARC874 (SEQ ID NO: 76) was the only other clone that bound to C5with a K_(d) of 1 uM.

The individual sequences listed in Table 6 are listed in the 5′ to 3′direction and were derived from aptamers that were selected under thedRmY SELEX conditions provided. In the embodiments of the inventionderived from this selection (and as reflected in the sequence listing)the purines (A and G) are deoxy and the pyrimidines (U and C) are2′-OMe. Each of the sequences listed in Table 6 may or may not containcapping (e.g., a 3′-inverted dT).

TABLE 6 Nucleotide sequences of ARC913 minimized clonesARC874 (SEQ ID NO: 76) CCUUGGUUUGGCACAGGCAUACAUACGCAGGGARC875 (SEQ ID NO: 77) CCUUGGUUUGGCACAGGCAUACAAACGCAGGGARC876 (SEQ ID NO: 78) GGGUUUGGCACAGGCAUACAUACCC ARC877 (SEQ ID NO: 79)GGGUUUGGCACAGGCAUACAAACCC ARC878 (SEQ ID NO: 80)GGCGGCACAGGCAUACAUACGCAGGGGUCGCC ARC954 (SEQ ID NO: 81)CGUUCUACCUUGGUUUGGCACAGGCAUACAUACGCAGGGGUCGAUCG

Example 3B: Optimization of ARC913: Doped Reselection

In order to both optimize clone ARC913 (SEQ ID NO: 75) for C5 bindingaffinity and to determine the key binding elements, a doped reselectionwas conducted. Doped reselections are used to explore the sequencerequirements within an active clone or minimer. Selections are carriedout with a synthetic, degenerate pool that has been designed based on asingle sequence. The level of degeneracy usually varies from 70% to 85%wild type nucleotide. In general, neutral mutations are observed but insome cases sequence changes can result in improvements in affinity. Thecomposite sequence information can then be used to identify the minimalbinding motif and aid in optimization efforts.

Pool preparation: The template sequenceTaatacgactcactataGGGAGAGGAGAGAACGTTCTACN₍₃₀₎GTTACGACTAGCATCGATG (SEQ IDNO: 82) was based on ARC913 (SEQ ID NO: 75) and was synthesized witheach residue originating from the random region doped at a 15% level,i.e. at each random (“N”) position, the residue has a 85% chance ofbeing the nucleotide found in the wild type sequenceCTTGGTTTGGCACAGGCATACATACGCAGGGGTCGATCG (SEQ ID NO: 83) and a 15% chanceof being one of the other three nucleotides.

The template and RNA pool for the doped reselection were preparedessentially as described above. The templates were amplified with theprimers taatacgactcactataGGGAGAGGAGAGAACGTTCTAC (SEQ ID NO: 84) andCATCGATGCTAGTCGTAAC (SEQ ID NO: 85). Two selections were done with fulllength C5, one selection using a higher concentration of salt in thewash step. The selection protocol was carried out as described above,with two exceptions: 1) Round 1 was done on hydrophobic plates (as wellas all subsequent rounds) with only a positive step; and 2) nocompetitor was used at all during the selection. The C5 concentrationand RNA pool concentration were kept constant at 200 nM and 1 uMrespectively.

Doped reselection data. Both the normal and high salt selections wereenriched after 5 rounds over the naïve pool. At round 5 the pool K_(d)was approximately 165 nM for the high salt selection and 175 nM for thenormal salt selection. The extent of binding was about 20% at theplateau in both. The R4 pools were cloned using TOPO TA cloning kit(Invitrogen, Carlsbad, Calif.), and 48 clones from each pool weresequenced. 12 clones from each pool were transcribed and assayed in asingle point dot blot assay at 500 nM C5. Dissociation constants(K_(d)s) were again measured using the dot blot assay previouslydescribed. K_(d)s were estimated for the 11 best clones identified inthe single point screen, by fitting the data to the equation: fractionRNA bound—amplitude*K_(d)/(K_(d)+[C5]). The clones with the three bestK_(d)s were SEQ ID NO: 91 (73 nM), SEQ ID NO: 96 (84 nM) and SEQ ID NO:95 (92 nM). The sequences for these 11 clones are listed below in Table7.

The sequences listed in Table 7 are listed in the 5′ to 3′ direction andrepresent the nucleotide sequences of the aptamers that were selectedunder the dRmY SELEX conditions provided. In the embodiments of theinvention derived from this selection (and as reflected in the sequencelisting), the corresponding sequences comprising the dRmY combinationsof residues, as indicated in the text, wherein the purines (A and G) aredeoxy and the pyrimidines (U and C) are 2′-OMe. Each of the sequenceslisted in Table 7 may or may not contain capping (e.g., a 3′-inverteddT). The unique sequences of each of aptamer below begins at nucleotide23, immediately following the 5′ fixed sequence GGGAGAGGAGAGAACGUUCUAC(SEQ ID NO: 86), and runs until it meets the 3′fixed nucleic acidsequence GUUACGACUAGCAUCGAUG (SEQ ID NO: 87).

TABLE 7 Nucleotide sequences of clones from doped reselection(SEQ ID NO: 88) GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUACAUACGCAGGGGUCGAUCGGUUACGACUAGCAUCGAUG (SEQ ID NO: 89)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUACAUACGCAGGUGUCGAUCUGUUACGACUAGCAUCGAUG (SEQ ID NO: 90)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUAAAUAGGCAGGGCUCGAUCGGUUACGACUAGCAUCGAUG (SEQ ID NO: 91)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCCCAGGCAUAUAUACGCAGGGAUUGAUCCGUUACGACUAGCAUCGAUG (SEQ ID NO: 92)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCGCAGGCAUACAUACGCAGGUCGAUCGGUUACGACUAGCAUCGAUG (SEQ ID NO: 93)GGGAGAGGAGAGAACGUUCUACCUUGUUGUGGCACAGCCAACCCUACGCACGGAUCGCCCGGUUACGACUAGCAUCGAUG (SEQ ID NO: 94)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUACAUACGCA GGUCGAUCGGUUACGACUA(SEQ ID NO: 95) GGGAGAGGAGAGAACGUUCUACCUUAGGUUCGCACUGUCAUACAUACACACGGGCAAUCGGUUACGACUAGCAUCGAUG (SEQ ID NO: 96)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCNCAGGCAUANAUACGCACGGGUCGAUCGGUUACGACUAGCAU (SEQ ID NO: 97)GGGAGAGGAGAGAACGUUCUACCUUUCUCUGCCACAAGCAUACCUUCGCGGGGUUCUAUUGGUUACGACUAGCAUCGAUG (SEQ ID NO: 98)GGGAGAGGAGAGAACGUUCUACCUUGGUUUGGCACAGGCAUAUAUACGCAGGGUCGAUCCGUUACGACUAGCAUCGAUG

Example 3C: 40 kDa Branched PEG Modification of ARC186

The oligonucleotide 5′NH₂-fCMGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfJUAfCfCfUmGfCmG-3T-3′ (ARC672, SEQ ID NO: 63) was synthesized on an ExpediteDNA synthesizer (ABI, Foster City, Calif.) according to the recommendedmanufacturer's procedures using standard commercially available 2′-OMeRNA and 2′-F RNA and TBDMS-protected RNA phosphoramidites (GlenResearch, Sterling, Va.) and a inverted deoxythymidine CPG support.Terminal amine function was attached with a 5′-amino-modifier,6-(Trifluoroacetylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,C6-TFA(Glen Research, Sterling, Va.). After deprotection, the oligonucleotideswere purified by ion exchange chromatography on Super Q 5PW (30) resin(Tosoh Biosciences) and ethanol precipitated.

The amine-modified aptamer was conjugated to different PEG moietiespost-synthetically. The aptamer was dissolved in a water/DMSO (1:1)solution to a concentration between 1.5 and 3 mM. Sodium carbonatebuffer, pH 8.5, was added to a final concentration of 100 mM, and theoligo was reacted overnight with a 1.7 molar excess of the desired PEGreagent (e.g. ARC1905 40 kDa Sunbright GL2-400NP p-nitrophenyl carbonateester [NOF Corp, Japan], or ARC187 40 kDa mPEG2-NHS ester [Nektar,Huntsville Ala.]) dissolved in an equal volume of acetonitrile. Theresulting products were purified by ion exchange chromatography on SuperQ 5PW (30) resin (Tosoh Biosciences), and desalted using reverse phasechromatography performed on Amberchrom CG300-S resin (Rohm and Haas),and lyophilized. The structure of ARC187 (SEQ ID NO: 5) is shown in FIG.21 while the structure of ARC1905 (SEQ ID NO: 67) is shown in FIG. 22.

Example 4 Isolated Perfused Heart Model Example 4A: Proof of Principlewith ARC186

The average concentration of complement component C5 in human plasma isapproximately 500 nM. Upon exposure of isolated mouse hearts perfusedwith Krebs Heinseleit buffer to 6% human plasma, the human complementcascade is activated, leading to cleavage of C5 into C5a and C5b.Component C5b subsequently forms a complex with complement componentsC6, C7, C8 and C9 also known as the “membrane attack complex” (“MAC” orC5b-9) which damages heart blood vessels and cardiac myocytes, thusleading to myocardial dysfunction (increased end diastolic pressure,arrhythmias) and asystole (Evans et. al., Molecular Immunology, 32,1183-1195 (1995)). Previously, monoclonal and single chain antibodiesthat block human C5 cleavage (Pexelizumab or a single-chain scFv versionof Pexelizumab) were tested in this model and shown to inhibitmyocardial damage and dysfunction (Evans et al, 1995).

This model was used to establish that the C5-blocking aptamer ARC186(SEQ ID NO: 4), like Pexeluzimab, inhibited human C5-mediated complementdamage to isolated perfused mouse hearts. C57 Bl/6 mice were purchasedfrom Charles River Laboratories, (Wilmington, Mass.). Briefly, followinginduction of deep anesthesia, each mouse heart was removed and mountedon a blunt needle inserted into the aorta, through which the heart wascontinuously perfused with Krebs Heinseleit buffer. A pressuretransducer (Mouse Specifics, Boston, Mass.) was inserted into the leftventricle allowing continuous measurement of the heart rate andintraventricular pressure. After a 15-minute period of equilibrationduring which baseline measurements were taken, hearts were subsequentlyperfused with buffer and 6% human plasma+/−aptamer at variousconcentrations (See FIG. 23). During these studies and as described inEvans et al., we demonstrated that hearts which were perfused with KrebsHeinseleit buffer+6% human plasma experienced failure within 5 minutesof adding the plasma to the perfusate, whereas hearts that werecontinuously perfused with buffer alone continued to beat in excess oftwo hours. Hence, the length of each experiment was arbitrarily definedas 15 minutes. The outline of this study with ARC186 is presented inFIG. 23.

Intraventricular pressure was monitored and recorded continuouslyresulting in a pressure wave tracing (FIG. 24 and FIG. 25). The lowestdeflection point represents the end diastolic pressure (“EDP”) and thehighest deflection point represents the systolic pressure (“SP”).Baseline pressure waves appear to the left of the vertical black linemarked “0” shown on each tracing. As previously published (Evans et al,1995), hearts perfused with 6% human plasma experienced a rapid increasein left ventricular end diastolic pressure, ultimately culminating inasystole (the heart stops) within 5 minutes (FIG. 24). When irrelevantaptamer was added to the human plasma at 50-fold molar excess, increasedEDP and asystole were also observed (FIG. 24).

When ARC186 was added to the system at molar equivalence, there was alsoa precipitous increase in EDP, culminating in asystole (FIG. 25). In allthree groups of hearts that experienced complement-mediated damage,increased EDP and asystole, the heart was visibly edematous and turgidby the end of the experiment. When ARC186 was added to plasma in 10-foldor 50-fold (FIG. 25) molar excess, ventricular pressure waves remainednormal and asystole was not observed. In addition, the previouslydescribed edema and turgidity were not apparent in these groups.

During each experiment, the heart rate was recorded at 5-minuteintervals, and the average heart rate for the group during each intervalwas graphed. As shown in FIG. 26 hearts perfused without aptamer or withirrelevant aptamer developed asystole quickly, usually within 5 minutes.ARC186 added to the system at molar equivalence slightly delayed theonset of asystole. Hearts in this group ultimately failed, however.ARC186 added to the plasma at 10-fold or 50-fold molar excess preservedthe heart rate for the duration of each experiment.

The percent increase in heart weight over baseline was calculated for arepresentative sample of failed hearts (no aptamer or 50-fold molarexcess of irrelevant aptamer) and compared to ARC186-protected hearts(10-fold and 50-fold molar excess of ARC 186). As shown in FIG. 27, ARC186 protected hearts gained significantly less weight than the failedhearts in the control groups.

Because ARC186 inhibits C5 but not C3 cleavage, C3 cleavage products(C3a) but not C5 cleavage products (C5a or C5b) should be found in theeffluent flowing from the isolated hearts protected by ARC186. Todirectly show that ARC186 inhibited cleavage of human plasma C5, therelative levels of human complement proteins C5a and C5b (C5 cleavageproducts) and C3a (a C3 cleavage product) were measured in the buffereffluent from the various groups by commercially available ELISA kits(C5b-9 ELISA kit, Quidel, San Diego, Calif.; C5a and C3a ELISA kit, BDBiosciences, San Diego, Calif.). ARC186 inhibited human plasma C5cleavage and the production of C5a (FIG. 28) and C5b-9 (FIG. 29) in adose-dependent manner. In contrast, ARC186 had no effect on cleavage ofhuman C3 into C3a and C3b (FIG. 30) further demonstrating the C5specificity of the molecule.

Once generated, complement C3b and C5b fragments are deposited locallyon tissues in the vicinity of the site of complement activation.Following completion of the experiments, mouse hearts were frozen in OCTmedia (Sakura Finetek, Torrance, Calif.), sectioned and then stainedusing standard immunohistochemistry for the presence of human C3b (cloneH11, Chemicon, Temecula, Calif.), human C5b-9 (clone aEl1, DAKO,Carpinteria, Calif.) or control mouse IgG (Vector Laboratories,Burlingame, Calif.). Results of the study are presented in FIG. 31.

As described in this study, the C5-blocking aptamer ARC186 was tested inan ex vivo model of complement component C5-mediated tissue damage whichuses isolated mouse hearts perfused with Krebs Heinseleit buffer and 6%heparinized human plasma, based on a model described in a previouslypublished study that tested the effects of the anti-C5 antibody,Pexeluzimab on the complement system (Evans, Molecular immunol 32:1183,(1995). Using this model, it was demonstrated that the C5—blockingaptamer (a) inhibited cleavage of human plasma C5 (but not C3), (b)inhibited deposition of human C5b (but not C3b) on mouse heart tissueand (c) inhibited human C5b-9 mediated myocardial dysfunction atclinically relevant concentrations (5 μM, a 10-fold molar excess ofaptamer vs. C5). These data show that when the human complement cascadeis activated in a physiologically relevant manner, C5-blocking aptamersare able to inhibit cleavage of plasma C5 and prevent myocardial damageand dysfunction.

Example 4B: Efficacy of PEGylated Aptamer

The material and methods used in this study were exactly the same asdescribed in Example 4A above. The experimental design and results arepresented in FIG. 32. The first half of the experiment used humanheparinized plasma (Center for Blood Research, Harvard Medical School,Boston, Mass.) as a source of complement and the second half usedheparinized cynomolgus macaque plasma (Charles River Laboratories,Wilmington, Mass.) as a source of complement. A PEGylated aptamer(ARC658; SEQ ID NO:62) was added to the system at increasing molarratios. Although all of the relevant ventricular pressure tracings werecollected, the table lists the presence or absence of an increase in enddiastolic pressure (EDP), whether or not asystole occurred and the timeuntil heart failure (defined as the presence of an elevated EDP andasystole).

During experiments with human plasma, the optimal dose of AR658 (SEQ IDNO: 62) was determined to be molar equivalence (500 nM) whereas duringexperiments with non-human primate plasma, a 50-fold molar excess (25μM) was necessary to protect the heart from C5b-mediated damage (seeFIG. 32).

These data are consistent with the difference in the affinity of theanti-C5 aptamer for human v. non-human primate C5 indicated by the invitro data. While not wishing to be bound by any theory, during oursubsequent cynomolgus macaque PK/PD studies described in Example 5, weadditionally demonstrated that a 30-fold molar excess of aptamer wasnecessary to inhibit zymosan-mediated plasma C5 cleavage, furthersupporting the notion that the aptamer binds primate C5 with loweraffinity than human C5.

Collectively, these studies indicate that both C5-blocking aptamersARC186 (SEQ ID NO: 4) and to a greater extent ARC658 (SEQ ID NO: 62) areefficacious in the mouse isolated, perfused heart model. This model alsodemonstrated that significantly more ARC658 (SEQ ID NO: 62) had to beused to inhibit cynomolgus macaque plasma C5-mediated heart damage (30+molar excess), compared with human C5-mediated heart damage (molarequivalence), further supporting in vitro data which indicated that theaptamer had lower affinity for primate C5. Finally, these data indicatedthat cynomolgus macaques would need to be dosed beyond a 30-fold molarexcess in order to demonstrate in vivo C5 blockade during PK/PD studies.

Example 5 Drug Metabolism & Pharmacokinetics of Anti-C5 Aptamers inAnimals

In Examples 5A-5G, all mass based concentration data refers only to themolecular weight of the oligonucleotide portion of the aptamer,irrespective of the mass conferred by PEG conjugation.

Example 5A: Metabolic Stability of the C5 Inhibitor ARC 186 in Primateand Rat Plasma

The non-PEGylated oligonucleotide precursor of the aptamers (i.e., ARC186; SEQ ID NO: 4) was tested in rat and cynomolgus macaque plasma(Charles River Labs, Wilmington, Mass.) in order to assess itsstability, rate kinetics, and pathways of degradation. Testing wasperformed using 5′ end-radiolabeled (³²P) aptamer incubated at 37° C. in95% pooled plasma (citrated) over the course of 50 hrs. At selected timepoints, aliquots of aptamer-containing plasma were withdrawn,immediately flash frozen in liquid nitrogen, and stored at −80° C.Detection and analysis of the aptamer and its metabolites in plasma wasaccomplished using liquid-liquid (phenol-chloroform) extraction followedby gel electrophoresis (on a 10% denaturing polyacrylamide sequencinggel) and high-resolution autoradiography.

FIG. 33 shows a log-linear plot of remaining percent of full-lengthaptamer as a function of incubation time in both rat and cynomolgusmacaque plasma. The degradation profile in both species appears to beessentially monophasic, with a rate constant of approximately k˜0.002hr⁻¹.

Example 5B: Pharmacokinetics of ARC657, ARC658 and ARC187 in the RatFollowing Intravenous Administration

To assess the pharmacokinetic profile of ARC657 (SEQ ID NO: 61), ARC658(SEQ ID NO: 62) and ARC187 (SEQ ID NO: 5), and to estimate the requireddosing level and frequency in primates and humans, a pharmacokineticstudy was conducted in catheterized Sprague-Dawley rats (Charles RiverLabs, Wilmington, Mass.). Aptamers were formulated for injection at 10mg/mL (oligo weight) in standard saline and sterile-filtered (0.2 m)into a pre-sterilized dosing vial under aseptic conditions. The route ofadministration used for the rat study was an intravenous bolus via thetail vein at a dose of 10 mg/kg. Study arms consisted of 3 animals pergroup, from which serial bleeds were taken pre-dose and at specifiedtime points over the course of 48 hours. The study design is outlined inFIG. 34. Blood samples were obtained from the surgically implantedjugular vein catheters, transferred directly to EDTA-coated tubes, mixedby inversion, and placed on ice until processing for plasma.

Plasma was harvested by centrifugation of blood-EDTA tubes at 5000 rpmfor 5 minutes and supernatant (plasma) was transferred to a freshpre-labeled tube. Plasma samples were stored at −80° C. until the timeof analysis. Analysis of plasma samples for ARC187 was accomplishedusing a homogeneous assay format utilizing the direct addition of plasmaaliquots to assay wells containing the commercially availablefluorescent nucleic acid detection reagent Oligreen™ (Molecular Probes,Eugene, Oreg.). After a brief incubation period (5 min) at roomtemperature, protected from light, the assay plates were read by afluorescence plate reader (SpectraMax Gemini XS, Molecular Devices,Sunnyvale, Calif.). The fluorescence signal from each well wasproportional to the concentration of aptamer in the well, and sampleconcentrations were calculated by interpolation of fluorescence valuesfrom a fluorescence-concentration standard curve (mean values fromduplicate or triplicate curves). Mean plasma concentrations wereobtained at each time point from the three animals in each group. Plasmaconcentration versus time data was subjected to noncompartmentalanalysis (NCA) using the industry standard pharmacokinetic modelingsoftware WinNonLin™ v.4.0 (Pharsight Corp., Mountain View, Calif.).Estimates were obtained for the following primary pharmacokineticparameters: maximum plasma concentration, C_(max); area under theconcentration-time curve, AUC; terminal half-life, t_(1/2); terminalclearance, C1; and volume of distribution at steady state, V_(ss).

Mean plasma concentration versus time data are shown in FIG. 35 and areplotted in FIG. 36. The concentration versus time data was subjected tononcompartmental analysis (NCA) using WinNonLin™ v.4.0. This analysisyielded the values presented in FIG. 37.

As anticipated, the 40 kDa aptamer ARC187 (SEQ ID NO: 5) had the longesthalf-life and the 20 kDa aptamer, ARC657 (SEQ ID NO: 61), the shortest.The observed Vss relative to the known plasma volume (˜40 mL/kg)suggested a moderate degree of binding/sequestration of ARC 187 (SEQ IDNO: 5) to proteins and/or tissue matrix in the extravascular space.Assuming a need to maintain a 5-fold molar excess of aptamer, theresults of this study suggested that ARC187 (SEQ ID NO: 5) provides asignificant advantage in terms of the dosing frequency and total amountof aptamer needed to maintain the desired plasma levels.

Previous studies (data not shown) in rodents and primates with aptamersof similar composition have shown dose proportionality/linearity atdoses up to 30 mg/kg, so it is not anticipated that this dosing levelwill result in nonlinear pharmacokinetic behavior.

Example 5C: Pharmacokinetics of ARC187 and ARC1905 in the MouseFollowing Intravenous Administration

To assess the pharmacokinetic profile of the ARC186 (SEQ ID NO: 4)oligonucleotide backbone conjugated to a different 40 kDa branched PEGthan that of ARC187 (SEQ ID NO:5), a pharmacokinetic study was conductedin female CD-1 mice (obtained from Charles River Labs, Wilmington,Mass.). Aptamers were formulated for injection at 2.5 mg/mL (oligoweight) in standard saline and sterile-filtered (0.2 m) into apre-sterilized dosing vial under aseptic conditions. The route ofadministration used for the mouse study was an intravenous bolus via thetail vein at a dose of 10 mg/kg. Study arms consisted of 3 animals pergroup, from which terminal bleeds were taken pre-dose (i.e., thenon-dosed control group) and at specified time points over the course of72 hours. The study design is outlined in FIG. 38A.

Blood samples were obtained by terminal cardiac puncture, transferreddirectly to EDTA-coated tubes, mixed by inversion, and placed on iceuntil processing for plasma. Plasma was harvested by centrifugation ofblood-EDTA tubes at 5000 rpm for 5 minutes and supernatant (plasma) wastransferred to a fresh pre-labeled tube. Plasma samples were stored at−80° C. until the time of analysis. Analysis of plasma samples forARC187 and 1905 was accomplished using a homogeneous assay formatutilizing the direct addition of plasma aliquots to assay wellscontaining the commercially available fluorescent nucleic acid detectionreagent Oligreen™ (Molecular Probes, Eugene, Oreg.). After a briefincubation period (5 min) at room temperature, protected from light, theassay plates were read by a fluorescence plate reader (SpectraMax GeminiXS, Molecular Devices, Sunnyvale, Calif.). The fluorescence signal fromeach well was proportional to the concentration of aptamer in the well,and sample concentrations were calculated by interpolation offluorescence values from a fluorescence-concentration standard curve(mean values from duplicate or triplicate curves). Mean plasmaconcentrations were obtained at each time point from the three animalsin each group. Plasma concentration versus time data was subjected tononcompartmental analysis (NCA) using the industry standardpharmacokinetic modeling software WinNonLin™ v.4.0 (Pharsight Corp.,Mountain View, Calif.). Estimates were obtained for the followingprimary pharmacokinetic parameters: maximum plasma concentration,C_(max); area under the concentration-time curve, AUC; terminalhalf-life, t_(1/2); terminal clearance, C1; and volume of distributionat steady state, V_(ss). Mean plasma concentration versus time data areplotted in FIG. 38B.

The concentration versus time data was subjected to noncompartmentalanalysis (NCA) using WinNonLin™ v.4.0. This analysis yielded the valuespresented in FIG. 38C. As anticipated, the 40 kDa PEGs from both vendorsshowed pharmacokinetic equivalence in mice.

The same plasma samples for ARC187 and 1905 used for the oligreenanalysis described directly above were analyzed using a validated highperformance liquid chromatography (HPLC) assay with UV detection

Mean plasma concentration values for ARC187 and ARC1905 were calculatedusing Microsoft Excel 2003. When plasma concentration values were belowthe LLOQ of the bioanalytical assay at pre-dose (time 0), a zero valuewas assigned. Values below the LLOQ from samples taken post-dose wereomitted from mean plasma concentration calculations. Mean plasmaconcentration data were used in a model-independent PK analysis usingWinNonlin, version 5.1 (Pharsight Corporation, Mountainview, Calif.).The area under the plasma concentration-time curve (AUC_(0-last)) wasestimated using the linear trapezoidal rule. For calculations, any valuethat was below the LLOQ of the assay, except the pre-dose sample, wasexcluded from calculations for PK parameter estimates. The apparentterminal half-life was calculated using the formula t_(1/2)=0.693/λ_(z)where λ_(z) is the elimination rate constant estimated from theregression of the terminal slope of the concentration versus time curve.At least three plasma concentration values after the peak concentrationon the terminal phase were used to determine λ_(z) and the coefficientof determination (r²) was required to be ≥0.85.

Overall, the HPLC analysis confirms the oligreen analysis describedimmediately above showing that ARC 1905 and ARC 187 were found to bebioequivalent based on comparisons of mean C_(max), AUC_(0-last) andAUC_(0-co) parameter estimates. Differences in AUC_(0-last) andAUC_(0-co) values for ARC1905 relative to ARC187 (as measured by HPLC)were well within bioequivalence acceptability criteria of ±20%.

Example 5D: Tissue Uptake Study of the C5 Inhibitors ARC657, ARC658 andARC187 in the Mouse Following Intravenous Bolus Administration

Female CD-1 mice were obtained from Charles River Labs (Wilmington,Mass.). Formulation of ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO: 62)and ARC187 (SEQ ID NO: 5) for injection was in saline at 5 mg/ml. Dosingformulations were sterile-filtered (0.2 μm) into pre-sterilized dosingvials under aseptic conditions and animals were given an intravenousbolus via the tail vein at a dose of 25 mg/kg. The study consisted ofgroups of 3 animals for each of four time-points, t=pre-dose, 3, 6, 12hrs. Following exsanguination, the vasculature of each animal wasflushed extensively (V˜30 mL) with saline to remove any blood left inthe vasculature. Tissues (heart, liver, kidney) were harvested, weighed,then homogenized at 50% w/v in standard saline, and stored at −80° C.until the time of analysis.

Analysis of tissue for ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO: 62),and ARC187 (SEQ ID NO: 5) was accomplished using a hybridization-basedELISA-type assay. In this assay, a biotinylated capture probe waspre-immobilized in the wells of a 96-well microplate at a bindingsolution concentration of 125 nM for 3 hrs. The plate wells were washed5 times with IX PBS. The plates were then blocked with 150 μl/well of a1× SuperBlock in TBS (Pierce Chemical, Rockford, Ill.). Plates werewashed again, covered, and stored at 4° C. until use. In separate tubes,the samples(s) were annealed in a buffer containing a FAM-labeled(5′-Fluorescein) sample-detection probe at 200 nM at 90° C. for 10 min,then quenched on ice. Concentration standards and control samples ofplasma/tissue were also pre-annealed with sample-detection probesolutions and then pipetted into assay plate wells containingimmobilized biotin capture probe, and annealed at 45° C. for 2.5 hrs.Plates were then washed again, and filled with 100 μl/well. of asolution containing 1×PBS containing 1 μg/mL of anti-fluoresceinmonoclonal antibody conjugated to horse radish peroxidase (anti-FITCMAb-HRP, Molecular Probes, Eugene, Oreg.) in 1×PBS, and incubated forapproximately 1 hr. Plates were washed again as above. Assay plate wellsare were then filled with 100 μl of a solution containing a fluorogenicHRP substrate (QuantaBlu, Pierce Chemical, Rockford, Ill.), andincubated for 20-30 min protected from light. After 45 minuteincubation, 100 μl/well of a stop solution was added to quench thefluorescent precipitate-producing reaction. Plates were read immediatelyon a fluorescence microplate reader (SpectraMax Gemini XS, MolecularDevices, Sunnyvale, Calif.) with fluorescence excitation at 325 nm andemission detected at 420 nm. Each well was read 10 times. All threeaptamers were detectable in the heart tissue at the three timepoints(FIG. 39).

Example 5E: Pharmacokinetics and Pharmacodynamics of the C5 InhibitorsARC657, ARC658 and ARC187 in the Cynomolgus Macaque FollowingIntravenous Administration Study 1

Formulation of ARC657 (SEQ ID NO: 61), ARC658 (SEQ ID NO: 62) and ARC187(SEQ ID NO: 5) for injection was in standard saline at 10 mg/mL anddosing formulations were sterile-filtered (0.2 μm) into pre-sterilizeddosing vials under aseptic conditions. The route of administration usedfor the macaque study was an intravenous bolus via a surgicallyimplanted femoral vein catheter at a dose of 30 mg/kg (approximately50-fold molar excess). The study design is outlined in FIG. 40. Bloodsamples were obtained from the femoral vein catheters, transferreddirectly to sodium citrate-coated tubes, mixed by inversion, and placedon ice until they were centrifuged to separate plasma (3000 rpm for 5minutes). Plasma was then divided into 250 μl aliquots which were storedat −80° C. and one aliquot of each sample was evaluated for aptamerconcentration using the fluorescence-based Oligreen™ assay previouslydescribed in the rat PK section above.

The primary plasma concentration versus time data is presented intabular form in FIG. 41. As anticipated, the 40 kDa PEG aptamer ARC187(SEQ ID NO: 5) persisted in plasma for the longest period of timewhereas the 20 kDa PEG aptamer ARC657 (SEQ ID NO: 61) persisted for theshortest amount of time. Inspection of the data shown in FIG. 41suggested that the data would best be fit by a two-compartment model.Thus, the pharmacokinetic parameter estimates reported in FIG. 42 werederived from the two-compartment model using the industry standardpharmacokinetic modeling software WinNonLin™ v.4.0 (Pharsight Corp.,Mountain View, Calif.).

As shown in FIG. 42, all of the aptamers had a similar Cmax value,between 23 and 30 μM, indicating that the aptamer dose (30 mg/kg) wassufficient to achieve a 50-fold molar excess of plasma aptamer vs C5concentration (50 fold molar excess, about 25 μM). Although they differby 10,000 molecular weight, ARC657 (20 kDa PEG) (SEQ ID NO: 61) andARC658 (30 kDa PEG) (SEQ ID NO: 62) had similar exposure (AUC),t_(1/2)(α) and t_(1/2) (β) values. In contrast, ARC187 (SEQ ID NO: 5)had significantly higher exposure (AUC) values, a prolonged t_(1/2) (α)and a slightly longer t_(1/2) (β) than the other molecules.

Additional aliquots of the plasma samples collected during thepharmacokinetics study were subsequently analyzed in vitro to determinethe efficacy of primate C5 blockade. The zymosan activation assay wasrun as described above to determine the amount of primate C5b-9 and C5a,generated, respectively. The data were plotted in several differentformats including C5b-9 concentration versus sample time (FIG. 43A),C5b-9 concentration versus aptamer concentration (FIG. 43B), C5aconcentration versus sample time (FIG. 43C), and C5a concentrationversus aptamer concentration (FIG. 43D).

The 40 kDa PEG aptamer ARC187 (SEQ ID NO: 5) inhibited primate C5cleavage (C5b-9 and C5a concentration) for the longest period of time(FIG. 43A and FIG. 43C). When the C5b-9 and C5a data were plotted versusaptamer concentration, it indicated that the concentration of C5blocking aptamer had to exceed 30-fold molar excess, regardless of thesize of the PEG molecules, in order for C5 cleavage to be completelyinhibited (FIG. 43B and FIG. 43D).

In summary, the data from the cynomolgus macaque PK/PD study demonstratethat (a) as anticipated, at least a 30-fold molar excess of aptamer(about 15 μM plasma aptamer concentration) was necessary to inhibit C5cleavage in vivo in the cynomolgus macaque, regardless of the size ofthe PEG group, (b) C5-blocking aptamers did not cause overt toxicity inthis species, and (c) when animals were dosed at a relatively highlevels (50-fold molar excess), plasma aptamer levels were well withinthe appropriate assay range during the period of sampling to allowcalculation of pharmacokinetic parameters

Example 5F: Pharmacokinetics and Pharmacodynamics of the C5 InhibitorsARC658 and ARC187 in the Cynomolgus Macaque Following IntravenousAdministration—Study 2

Study 2 was similar in design to study 1 described above, with thefollowing exceptions a) only two compounds were evaluated (ARC658 (SEQID NO: 62) and ARC187 (SEQ ID NO: 5); b) the number of animals wasincreased to four per group; and c) the 1-minute plasma sample wasdeleted and replaced with a 144 hour sample to ensure that the terminalhalf-life calculation was based upon more data points. The formulationand dosing of these two aptamers, blood sampling and plasma isolationtechniques was identical to the methods described above in study 1. Thedesign for study 2 is summarized in FIG. 44.

Following completion of study 2, plasma aliquots were analyzed asdescribed in study 1 to determine the a) the concentration of aptamer inplasma at various timepoints following intravenous administration, andb) the efficacy of C5 blockade.

Plasma aptamer concentration was plotted as a function of time (FIG. 45)and the primary data for ARC658 (SEQ ID NO: 62) and ARC187 (SEQ ID NO:5) are presented in tabular form in FIG. 39 and FIG. 40, respectively.The 40 kDa PEG aptamer ARC187 (SEQ ID NO: 5) persisted in plasma for thelongest period of time. Inspection of FIG. 45 indicated that the datawould be best fit by a two-compartment model. Thus, the pharmacokineticparameter estimates reported in FIG. 46 were derived from thetwo-compartment model using WinNonLin™ v.4.0 (Pharsight Corp., MountainView, Calif.).

Comparing the pharmacokinetic parameters generated during the PK/PDstudy 1 and study 2 above, the data for ARC658 (SEQ ID NO: 62) andARC187 (SEQ ID NO: 5) were similar with the exception of the t_(1/2)(α)measurement for ARC187. While not wishing to be bound by any theory, thediscrepancy in the t_(1/2)(α) measurements for ARC 187 between the twostudies is likely due to the small sample size in the pilot study.

As demonstrated in FIG. 46, the Cmax values were similar for ARC658 (SEQID NO: 62) and ARC 187 (SEQ ID NO: 5). In contrast, drug exposure (AUC)was significantly greater in animals treated with ARC187 (SEQ ID NO: 5).Also, ARC187 had prolonged t_(1/2)(α) and t_(1/2)(β) values as comparedto ARC658 (SEQ ID NO: 62). These data, along with the data generatedduring the PK/PD study 1 indicate that of the C5-blocking aptamersARC187 may provide the most effective in vivo C5 blockade for a givendose.

Additional aliquots of the plasma samples collected during thepharmacokinetics study were subsequently analyzed in vitro to determinethe efficacy of primate C5 blockade. As before, the zymosan activationassay was run to determine the amount of primate C5b-9 and C5a,respectively, generated. The data were plotted as C5b-9 concentrationversus aptamer concentration (FIG. 47) and C5a concentration versusaptamer concentration (FIG. 48). As previously demonstrated during PK/PDstudy 1, the concentration of C5 blocking aptamer must exceed a 30-foldmolar excess (aptamer to plasma C5 concentration), or approximately 15μM, regardless of the size of the PEG molecule, in order for primate C5cleavage to be completely inhibited (FIG. 41 and FIG. 42).

By inspecting the data in the tables of FIG. 39 and FIG. 40, it isapparent that after a 30-mg/kg I.V. bolus, ARC658 (SEQ ID NO: 62)remains above 15 μM for approximately 4 hours whereas ARC 187 remainsabove 15 μM for approximately 8 hours. Thus, given a similar dose ofdrug, the 40 K aptamer ARC187 provides clinical efficacy forapproximately twice as long as the 30K aptamer ARC658 (SEQ ID NO: 62).

In summary, cynomolgus macaques must be treated with at least a 30-foldmolar excess of aptamer vs plasma C5 in order to block C5 conversion invivo. These data are consistent with previous in vitro (hemolysis) andex-vivo (isolated perfused mouse heart) studies which suggested that theC5-binding aptamers had a lower affinity for primate C5 versus human C5.It has been shown that C5-blocking aptamers can safely be delivered asan intravenous bolus at a dose of up to 30 mg/kg, which equates toapproximately a 50-fold molar excess of aptamer vs C5 concentration.

Example 5G: ARC1905 in the Cynomolgus Macaque Following Bolus IVAdministration

The pharmacodynamics of the C5 inhibitors ARC1905 was evaluated in thecynomolgus macaque following intravenous administration. Formulation ofARC1905 for injection was in standard saline at 7.5 mg/mL and dosingformulations were sterile-filtered (0.2 μm) into pre-sterilized dosingvials under aseptic conditions. Cynomolgus monkeys (n=4) were dosed at 0(saline control) or 30 mg/kg via intravenous bolus administration. Bloodsamples were obtained from a peripheral vein or the arterial access portand blood samples (0.5 mL) were transferred into dipotassium (K₂) EDTAtubes, placed on wet ice, and centrifuged within 30 minutes ofcollection at approximately 4° C.

The plasma samples were analyzed in vitro to determine the efficacy ofARC1905 in primate C5 blockade. The zymosan assay previously describedwith respect to ARC 1905 in Example 1C was used to determine the amountof primate C5a generated. The decrease in post-zymosan C5a values at 0.5and 2 hours after dosing indicates that ARC1905 inhibits C5 cleavage invivo in the cynomolgus macaque in a similar manner as ARC 187 when dosedat approximately the same concentration and the same route ofadministration as measured in vitro using the zymosan activation assay.

Example 5H: Pharmacokinetics and Pharmacodynamics of the C5 InhibitorARC187 in the Cynomolgus Macaque Following Bolus IV Administration andInfusion

The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of ARC 187(SEQ ID NO: 5) were also evaluated in cynomolgus macaques after anintravenous loading bolus followed immediately by the initiation of anintravenous infusion. This study design is shown in FIG. 49.

The loading bolus dose and infusion rate necessary to achieve the targetsteady state plasma concentration of 1 uM were calculated using thepharmacokinetic parameters derived from the IV bolus—only study listedin FIG. 50.

A total of three cynomolgus macaques were administered an IV bolus ofARC187 at 1 mg/kg, followed immediately by the initiation of an IVinfusion at a rate of 0.0013 mg/kg/min for a period of 48 hrs. Samplesof whole blood were collected from 0 to 192 hours post-treatment, storedon wet ice, processed for plasma, and then stored frozen at −80 C. Theconcentration of ARC 187 in plasma samples was determined using both afluorescent nucleic acid stain assay (described in Example 5B) and aGLP-validated performance liquid chromatography (HPLC) assay. The HPLCassay method for the determination of ARC 187 in monkey plasma wasvalidated by ClinTrials Bio-Research (Montreal, Canada). The validationstudy complied with the United States Food and Drug Administration (FDA)Good Laboratory Practice (GLP) regulations (21 CFR § 58). The HPLC assaymethod was validated with respect to: selectivity, linearity, lowerlimit of quantitation (LLOQ), carry-over, intra-assay precision andaccuracy, inter-assay precision and accuracy, stock solution stability,injection medium stability, short-term matrix stability, freeze-thawstability, long-term matrix stability and dilution integrity. The usablelinear dynamic concentration range of assay was determined to be 0.080to 50.0 μM.

The measured PK profile of ARC187 under these conditions conformed wellto the calculated profile generated using only the IV bolus PKparameters (see FIG. 51). The target plasma concentration of 1 uM wasestablished in <5 min post-dose and maintained for the entire durationof infusion. After cessation of the infusion, the aptamer showed aterminal clearance half-life, t_(1/2)(β)˜40-60 hr.

The pharmacodynamic activity of ARC187 (SEQ ID NO: 5) in the cynomolgusmacaque was evaluated ex-vivo by using plasma samples collected duringPK study in the zymosan activation assay previously described with themodification that cynomolgus sample plasma was diluted 10-fold into 10%human plasma and then treated with 5 mg/mL zymosan. C5 activation, asreflected by the appearance of the C5a cleavage product, was measured byELISA specific to human C5a (C5a ELISA kit, BD Biosciences, San Diego,Calif.). The concentration of active ARC187 in each sample was thenquantified by comparison with a standard curve derived from zymosanassays using samples prepared with known ARC187 levels (see FIG. 52).This study indicates that ARC187 maintains its anti-complement activitythroughout the duration of and following infusion, at levelssubstantially consistent with the pharmacokinetic profile describedabove.

Example 5I: Prediction of Human Dosing Requirement

Human dosing requirements for prevention, amelioration, or treatment ofcomplications related to CABG surgery are based on the followingassumptions: first, CABG patients will be administered a singleintravenous bolus dose of the anti-C5 aptamer prior to initiatingsurgery, followed by continuous infusion to establish and maintain asteady-state plasma concentration of 1.5 μM for 24-48 hours post CABGsurgery. The bolus dose and infusion rate estimates are based uponcalculations using the pharmacokinetic parameters derived from thepreviously described IV bolus—only and bolus plus infusion studies incynomolgus macaques. The estimated bolus dose of ARC187 is 1 mg/kg, andthe associated infusion rate is 0.0013 mg/kg/min. For this bolus plus 48hr infusion regimen, the anticipated total drug requirement is 0.4 g forARC187, where mass refers to oligonucleotide weight only (see column 7in the table of FIG. 53). Column 2 of the table shown in FIG. 53 refersto the weight of the PEG group conjugated to oligonucleotide portion ofARC187, column three refers to the molecular weight of theoligonucleotide portion of ARC187 (and will be the same for all aptamersherein that comprise ARC186 (SEQ ID NO: 4) as its oligonucleotidesequence), column 4 refers to the molecular weight of 40 kDA PEGconjugated to ARC186 (SEQ ID NO: 4) via amine reactive chemistry asdescribed in Example 3C above, column 5 refers to ARC187's α phase halflife in a two compartment model, and column six refers to ARC187's βphase half life in a two compartment model.

Example 6 Anti-C5 Aptamers and Heparin/Protamine Interaction

One anticipated application of the anti-C5 aptamer is as a prophylacticfor the prevention or mitigation of inflammatory side effects associatedwith coronary artery bypass graft (CABG) surgery. High concentrations ofthe anticoagulant heparin (3-5 units/mL or 1-2 μM) are typicallyadministered during CABG to prevent thrombosis and maintain patencywithin components of the bypass pump; reversal of heparin's effect afterthe procedure, and restoration of normal hemostasis, is achieved by theadministration of similarly high concentrations of protamine (˜5 μM).Given the potential dangers to patients of any interference in theeffectiveness of either of these drugs, it was necessary to demonstratethat anti-C5 aptamers (1) do not alter the activities of either drug and(2) do not display inherent effects on hemostasis that could complicatepatient anticoagulation treatment.

Heparin is a sulfated polysaccharide with a net negative charge and amean molecular mass of approximately 15 kDa that exerts an inhibitoryeffect on a number of proteases in the coagulation cascade by promotinginteractions with antithrombin. Protamine, a highly positively chargedpolypeptide, is able to block heparin activity via a poorlycharacterized interaction that is at least partially electrostatic innature. The functional core of ARC187 (SEQ ID NO: 5), like heparin, ishighly anionic. Thus, it is conceivable that ARC187 couldnonspecifically bind to heparin-binding sites or protamine and interferewith the activities of these molecules. The following studiesinvestigated the inherent (i.e., heparin-like) anticoagulant propertiesof ARC187, the effects of ARC187 on heparin function, the effects of ARC187 on heparin-neutralization by protamine, and the effects of protamineon the complement inhibiting properties of ARC187.

Example 6A: In Vitro Effects of ARC 187 on Coagulation

The inherent effects of ARC 187 (SEQ ID NO: 5) on plasma coagulabilitywere investigated using standard clinical tests of the extrinsic andintrinsic arms of the coagulation cascade, the prothrombin time (PT) andactivated partial thromboplastin time (aPTT), respectively. As shown inFIG. 54, titration of citrated human plasma with concentrations well inexcess of projected doses (up to 20 μM) resulted in no change in the PT,and only a slight elevation in the aPTT.

To assess the in vitro effects of ARC 187 on heparin and protaminefunctions, blood from 3 individuals was drawn into 4-5 units/mL heparin,doses associated with heparin levels used in CABG surgery. Thecoagulability of these samples was assessed using the activated clottime (ACT), a whole blood coagulation test routinely used to monitorheparin activity during surgery. At these concentrations of heparin, inthe absence of other additives, the ACT was significantly prolonged froma baseline value of ˜150 seconds to ˜500 seconds in the presence of 4U/mL heparin or ˜800 seconds in the presence of 5 U/mL heparin. Additionof 10 μM ARC187 to these samples had little effect on clot time,demonstrating that ARC 187 does not interfere with the anticoagulantactivity of heparin.

The heparin anticoagulant effect was readily neutralized by titrationwith protamine up to 6-8 μM (4 U/mL heparin) or 12 μM (5 U/mL heparin).ACT values in the presence of heparin and neutralizing concentrations ofprotamine were essentially indistinguishable from baseline. Since thenucleic acid core of ARC 187 (12 kDa) is of larger molecular weight thanprotamine (5 kDa), one might expect that equimolar concentrations ofARC187 added to protamine would be sufficient to completely reverse theneutralizing activity of protamine. However, preincubation of protaminewith approximately equivalent concentrations of ARC 187 had littleeffect on the ACT. Blood samples containing neutralizing concentrationsof protamine displayed similar ACT values in the presence or absence of10 μM ARC187, indicating that ARC187 has only a slight if any effect onthe procoagulant activity of protamine. These results are summarized inFIG. 55.

Example 6B: In Vivo Effects of ARC187 on Coagulation

The interactions between the function of heparin and protamine duringconcurrent administration of anti-C5 aptamer ARC187 (SEQ ID NO: 5), atclinical doses of heparin and clinical/subclinical/superclinical dosesof protamine were investigated to determine whether the presence ofsubclinical/superclinical plasma concentrations of ARC187 wouldinterfere with the reversal of heparin anticoagulation by protamine. Theresults of the study are summarized in FIG. 56. Briefly, the baselineACT values were unaffected by 10 uM (i.e., 10-fold molar excess of theclinical dose) of ARC187 at all heparin doses tested. Similarly, theextent of anticoagulation by heparin was unaffected by 10 uM ARC187. Inthe absence of ARC187, the minimum efficacious dose of protamine was˜30% (clinical dose=100%). Furthermore, the reversal of heparinanticoagulation by 30% protamine was unaffected by 10-fold molar excessof the clinical dose (i.e., 10 uM) of ARC187. Thus, the use of ARC187for complement inhibition in a clinical setting (e.g., CABG) should beunaffected by concurrent use of heparin and protamine at typical doses.

Example 6C: Effect of Heparin and Protamine on ARC187 Anti-ComplementFunction

The effects of heparin and protamine on the anti-complement activity ofARC187 (SEQ ID NO: 5) were examined in citrated whole blood samplesactivated with zymosan, as described in Example 1. Just prior to zymosanactivation, ARC187 was titrated into samples of citrated blood treatedunder four conditions: 1) no treatment (no heparin or protamine); 2) 4U/mL heparin; 3) 6 μM protamine; 4) 4 U/mL heparin+6 μM protamine.Following activation with zymosan, C5 activation was quantified by ELISAmeasurement of sC5b-9 in plasma (C5b-9 ELISA kit, Quidel, San Diego,Calif.). For each condition, the results, expressed as percentinhibition of C5 activation versus ARC 187 concentration, wereindistinguishable within error (see FIG. 57). In all cases completeinhibition was achieved with 1-2 μM ARC187. Thus, heparin and protamine,separately or combined at concentrations relevant to their use in CABGsurgery, do not appear to affect the anti-complement activity of ARC187.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the descriptionand examples above are for purposes of illustration and not limitationof the following claims.

1. A compound or salt thereof, wherein the compound comprises thesequence fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCfUmGmAmGfUfUfUAfCfCfUmGfCmG-3T (SEQ ID NO:4) and1,3-bis(mPEG)-propyl-2-(4-butamide); wherein fC and fU=2′-fluoronucleotides, and mG and mA=2′-OMe nucleotides and all other nucleotidesare 2′-OH and 3T indicates an inverted deoxy thymidine; and wherein thecompound binds specifically to complement system protein C5. 2-10.(canceled)
 11. A compound of claim 1, wherein the mPEG has a molecularweight greater than 10 kDA.
 12. A pharmaceutical composition comprisinga therapeutically effective amount of the compound of claim 1 and apharmaceutically acceptable carrier or vehicle.
 13. A method fortreating a C5 complement protein, C5a and/or C5b-9-mediated disorder,the method comprising administering to a patient in need thereof atherapeutically effective amount of the compound or pharmaceuticallyacceptable salt of the compound of claim
 1. 14. The method of claim 13,wherein the disorder is myocardial injury relating to CABG surgery,myocardial injury relating to balloon angioplasty, myocardial injuryrelating to restenosis, C5, C5a and/or C5b-9-complement protein mediatedcomplications relating to CABG surgery, percutaneous coronaryintervention, paroxysomal nocturnal hemoglobinuria, acute transplantrejection, hyperacute transplant rejection, subacute transplantrejection, or chronic transplant rejection.
 15. The method of claim 13,wherein the disorder is complement mediated ocular tissue damage.
 16. Apharmaceutical composition comprising a therapeutically effective amountof the compound of claim 11 and a pharmaceutically acceptable carrier orvehicle.
 17. A method for treating a C5 complement protein, C5a and/orC5b-9-mediated disorder, the method comprising administering to apatient in need thereof a therapeutically effective amount of thecompound or pharmaceutically acceptable salt of the compound of claim11.
 18. The method of claim 17, wherein the disorder is myocardialinjury relating to CABG surgery, myocardial injury relating to balloonangioplasty, myocardial injury relating to restenosis, C5, C5a and/orC5b-9-complement protein mediated complications relating to CABGsurgery, percutaneous coronary intervention, paroxysomal nocturnalhemoglobinuria, acute transplant rejection, hyperacute transplantrejection, subacute transplant rejection, or chronic transplantrejection.
 19. The method of claim 17, wherein the disorder iscomplement-mediated ocular tissue damage.